FINAL YEAR PROJECT
submitted in part fulfilment for the degree of
BSc (Hons) Biology
Name : Božena Pálinkášová
Title : Investigations into the Lambda / HindIII Restriction Fragment
Insertion Frequencies Using pUC18 as a Cloning Vector
Supervisor: Mr H N Hughes
Year : 2006 / 2007
SCHOOL OF BIOLOGY, CHEMISTRY AND HEALTH SCIENCE
FACULTY OF SCIENCE AND ENGINEERING
Abstract
Plasmids are essential tools in the molecular biology due to having a number of
advantageous properties. They are easy to manipulate and their covalent closed
circular form makes them easy to differentiate and purify from the linear genomic
DNA. They are extremely useful in molecular cloning, as they replicate independently
and produce up to 500 copies per cell.
In this project, frequencies of the lambda DNA / HindIII restriction fragments cloned
into pUC18 were investigated. Eight lambda DNA restriction fragments produced by
HindIII vary in size, ranging from 125bp – 23,130bp; therefore comparison of these
fragments of different sizes can be made. The plasmid was cut with HindIII creating a
complementary ‘sticky ends’, ligated at random with a mix containing eight lambda
fragments and transformed to Escherichia coli. Cells were allowed to replicate and
produce clones of the recombinant plasmid. After incubation for 24 hours, the
recombinant plasmid DNA was extracted and analysed.
Results showed that half of the lambda fragments failed to be inserted and cloned into
the pUC18, which was probably due to being considerably larger in comparison with
the size of the vector. The remaining fragments that were successfully cloned were
statistically analysed and the frequencies of the occurance were highly significantly
different.
During the project, the actual number of lambda restriction fragments produced by
HindIII was investigated due to contradictory literature statements. It was revealed
2
that wild-type lambda DNA has six restriction sites for HindIII and a standard lambda
laboratory strain (cIind 1 ts857Sam7), which used for this experiment, has seven
HindIII restriction sites due to a single base substitution.
3
Index
1. Introduction………………………………………………………………….....5-10
2. Aims and Objectives……………………………………………………………...11
3. Materials and Methods……………………………………………………….12-20
3.1. Preparation of the Vector and Ligation……………………………..….12-13
3.1.1. pUC18………………………………………………………………………....12
3.1.2. pMAL –c2X…………………………………………………………….....12-13
3.2. Transformation…………………………………………………….……….14-16
3.2.1. Selective Media………………………………………………………...……..14
3.2.2. Transformation Reaction……………………………………………...…15-16
3.3. Purification of Recombinant Plasmid…………………………………......16-18
3.4. Plasmid DNA Digestion and Analysis by Agarose Gel Electrophoresis...18-19
3.4.1. Plasmid DNA digestion with HindIII………………………………………..18
3.4.2. Analysis by Agarose Gel Electrophoresis……………………………….18-19
3.5. Lambda DNA / HindIII Restriction Fragments………………………......19-20
4. Results…………………………………………………………………………21-24
4.1. pUC18………………………………………………………………………..21-23
4.2. pMAL – c2X…………………………………………………………………….23
4.3. Lambda DNA / HindIII Restriction Fragments…………………..………….24
5. Discussion………………………………………………………..…………….25-29
6. Acknowledgements…………………………………………………………….....30
7. References……………………………………………………………………..31-35
Appendix I…………………………………………………………………….....36-49
Appendix II……………………………………………………………………...50-54
4
1. Introduction
In vivo cloning using living bacterial cells was one of the first methods used for DNA
amplification. The discovery of the existence of extrachromosomal circular DNA
molecules, plasmids, has contributed greatly to the development of recombinant
technology. One of the advantages of plasmids is that they can replicate independently
and produce a significant number of copies, ranging from a single copy to several
hundreds, within a single bacterial cell. The number of copies per cell depends on the
“plasmid-encoded control elements that regulate the initiation”, and the rate of the
replication of a plasmid (Del Solar et al. 1998). These naturally occurring double
stranded DNA molecules usually give a specific advantage to the host cell. Cells may
benefit from an antibiotic resistance gene carried by the plasmid or the ability to
metabolise substances other than glucose for a carbon source, but some plasmids may
also encode genes for virulence. Studies of plasmids have also led to the construction
of synthetic plasmids that can be designed and used for a number of specific purposes
(Viera and Messing 1991). Nowadays plasmids tend to be used as cloning vehicles to
amplify DNA segments of interest, to express genes coding for a protein product, to
sequence or even mutate genes. Synthetically derived plasmids are advantageous as
they can be designed to carry a selective marker that would, under the specific
conditions, differentiate cells that carry recombinant plasmid from cells that do not
carry it.
In this project an example of the synthetically derived plasmid, pUC18 (Figure 1), has
been used for in vivo cloning of lambda phage DNA fragments. The pUC18 vector
was originally derived from the plasmid pBR322, containing an ampicillin resistance
5
gene, which was fused with the E. coli
lacZ’ gene containing an introduced
multiple cloning site (Norrander et al.
1983 and Yanish-Perron et al. 1985). The
lacZ’ gene consists of the promoter,
operator and the first 146 codons that
code for the part of the amino acid
sequence of the product β-galactosidase.
Figure 1: Map of pUC18 plasmid vector
The rest of the sequence is complemented
(Adapted from: Fermentas, 2006b)
(α - complementation) by the E. coli host that has a partly deleted version of the gene,
lacZ ∆M15 (Brown, 2000). This small plasmid vector has 2686 base pairs (bp) and a
high copy number, with 500-700 copies per cell. The small size and high copy
number makes it extremely advantageous for cloning as it is easily taken up by the
host cell and produces a high number of target DNA (Parke, 1990). Also, a functional
lacZ’ gene within the pUC18 enables scientists to distinguish recombinant colonies
from non-recombinants by simple colour selection on medium containing the lactose
analogue, 5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside (X-Gal). The enzyme
product of the lacZ gene, β-galactosidase, hydrolyses X-Gal producing a coloured
substance that stains colonies that carry the plasmid blue (Messing, 1991). However,
if a DNA fragment is cloned into the polylinker site located within the lacZ, it disrupts
the gene resulting in white colonies (ibid). The ampicillin resistance gene in pUC18
also aids to selection of recombinants as only the colonies carrying the plasmid are
selected from untransformed colonies that would also appear to be white. The
resistance gene encodes β-lactamase that hydrolyses ampicillin in the selective
medium; hence the phenotype of the recombinants is ampr lacZ-. However,
6
occasionally small “satellite colonies” could appear in the close proximity of
recombinant colonies due to the degradation of ampicillin by β-lactamase, produced
by recombinants, in the nutrient agar. Subculturing the colonies into liquid media
containing ampicillin enables the selection of purely recombinant colonies (Sambrook
and Russel 2001a).
Other essential tools in recombinant DNA technology include restriction
endonucleases. Restriction endonucleases are enzymes isolated from microorganisms,
and their primary function is to protect the organism’s DNA from foreign viral DNA
that can invade the cell. Restriction endonucleases function by cleaving the phosphodiester bonds at specific recognition sites, hence degrading the foreign DNA
(Takasaki, 1994). Restriction enzymes fall into three main groups, Type I, II and III
that are further subdivided. Type I comprises a multisubunit protein complexes that
require ATP to function. They nick the DNA sequences at inconsistent sites (Roberts
et al. 2003). Type II restriction enzymes recognise DNA at specific sequences and
require metal ions (Mg2+) as co-factors to function (Cowan, 2004). They cleave at or
adjacent to the palindromic sequences, cleaving both DNA strands and producing free
5’ phosphate and 3’ hydroxyl groups (Smith and Wilcox 1970). Type III, composed of
two subunits, has an absolute requirement for ATP, as Type I, but cleaves at a specific
distance from their recognition sequence. In addition, recently a new category of
restriction enzymes was recognised as Type IV that cleaves methylated DNA
sequences (ibid). In this project a type II restriction endonuclease HindIII (isolated
from Haemophilus influenzae, serotype d) was used. This enzyme recognises a
specific palindromic sequence and cleaves the DNA of both complementary strands
between the two adenines of 5’ A↓AGCTT 3’ nucleotide sequence (Roberts, 1979).
7
Cleavage by the enzyme produces complementary sticky ends and therefore any DNA
fragments cleaved with HindIII can be ligated together to form a recombinant
molecule.
In this project, E. coli bacteriophage lambda was cut with HindIII to produce
restriction fragments for in vivo cloning into pUC18. Lambda phage DNA is 48,502bp
double stranded linear molecule with 12 single stranded complementary bases
extending in 5’ direction from the ends of a molecule (Chauthaiwale et al. 1992) and
seven restriction sites for HindIII (Fermentas, 2006a). Therefore, lambda DNA is
cleaved into eight restriction fragments: 23,130bp; 2,027bp; 2,322bp; 9,416bp; 560bp;
125bp; 6,557bp and 4,361bp. However, it was noted during the literature search that a
number of sources (Roberts, 1979; Roberts and Macelis 2007; Sambrook and Russel
2001a) state that lambda DNA had only six HindIII restriction sites resulting in seven
fragments (with no 125bp and 6,557bp fragments, but a single 6,682bp fragment).
Therefore, the above aspect was also investigated as a part of this project. The pUC18
was cut with HindIII at a single restriction site, which is located in the multiple
cloning site (MCS), disrupting the lacZ’ gene. Both, digested lambda DNA and
pUC18 were ligated together using DNA ligase by restructuring the phosphodiester
bond to produce recombinant molecules (Higgins and Cozzarelli 1979). The ligated
mix was then transferred into competent E. coli cells via transformation.
Transformation was chemically induced with CaCl2 and heat shocking the cells
(Chung and Miller 1993). The transformed mix was then grown on nutrient agar
plates containing X-Gal, lac operon inducer isopropyl-β-D-thiogalactopyranoside
(IPTG) and ampicillin. Plasmids carrying the lambda phage insert (white) were
harvested from the recombinants via an alkaline lysis method. Isolates were digested
8
with HindIII and the restriction fragments (plasmid and lambda DNA insert) were
separated using agarose gel electrophoresis to confirm the transformants and
determine the size of the lambda insert.
In this project, 121 isolated plasmids from the transformed E. coli colonies were
analysed. The insertion of lambda phage fragments cut with HindIII was studied to
determine the size and the frequency of the all possible inserts. In addition, the
intention of this study was to identify the maximum size of lambda phage fragment
that can be inserted into the pUC18. This plasmid vector is relatively small in size in
comparison with half of the cut lambda fragments, which are considerably larger.
Therefore, in this study it was investigated whether any of the larger fragments would
insert into this comparatively smaller plasmid vector.
For comparison with pUC18, the larger plasmid pMAL – c2x that has 6,648bp was
used (Brown, 1998). This vector is usually used for protein expression and has a
structural gene for maltose-binding protein (MBP) that fuses with the target protein
product of the inserted gene and is produced in the cytoplasm of the host cell (Di
Guan et al. 1988). The MBP is then used for the recombinant protein purification
(Maina et al. 1988). The vector is a synthetic derivative of pUC18 and has lacZ’ gene
with MCS and gene for ampicillin resistance (New England BioLabs, 2006).
Therefore, the recombinants can be easily screened for on X-Gal, IPTG and ampicillin
containing selective agar plates similarly to the pUC18 recombinants. Another
property of this vector is that it has a single HindIII restriction site within the MCS,
9
which allows a direct comparison of the frequencies of integrated HindIII cut lambda
DNA fragments with the frequencies of those that integrated into the pUC18.
10
2. Aims and Objectives:
The aim of this project was to clone lambda phage fragments in Escherichia coli
using pUC18 plasmid vector and compare cloned fragment frequencies.
The objective was to test the frequency of insertion of lambda phage DNA fragments
and test the following hypotheses.
Hypothesis: There will be a difference between the frequencies of the eight lambda
phage fragments that have been inserted into the plasmid and taken up by the E. coli
competent cells.
Null hypothesis: There will not be a difference between the frequencies of the eight
lambda phage fragments that have been inserted into the plasmid and taken up by the
E. coli competent cells.
11
3. Materials and Methods
3.1. Preparation of the Vector and Ligation
3.1.1. pUC18
A ligation mix containing 500ng of pUC18 plasmid (Sigma) and 1μg Lambda DNA
HindIII fragments (Invitrogen) in total volume of 50μl was provided for this study
(provided by MMU laboratory technician Mrs Noshina Shaheen).
3.1.2. pMAL - c2x
Phagemid vector pMAL - c2x was purchased from New England BioLabs. Vector
was digested with FastDigestTM HindIII (Fermentas) in 1.5ml sterile Eppendorf tubes,
so that 2μg of pMAL - c2x was digested with 20Units of enzyme. The enzyme buffer
10x FastDigestTM buffer (Fermentas) made 10 per cent of 50μl total volume reaction
mixture in double deionised water. The vector was digested at 37oC (water bath) for
one hour, centifuging the tubes in the bench top micro-centrifuge at 13,000rpm for
30seconds after every 30minutes to overcome any effects of condensation.
The vector was dephosphorylated with calf intestinal alkaline phosphatase (CIAP,
New England BioLabs). In this process the removal of 5’ phosphate groups from the
vector was catalysed by the alkaline phosphatase. The treatment usually helps to avoid
the self ligation of the vector as the two ends cannot be joined with DNA ligase that
has an absolute requirement for a free 5’ phosphate group (Powell and Gannon 2000).
12
Two units of alkaline phosphatase in 1x NEB Buffer3 (New England BioLabs) were
used to dephosphorylate the digested 2 μg of pMAL - c2x. Samples were incubated at
37oC (water bath) for 30 minutes. Samples were microcentrifuged at 13000rpm for 30
seconds at the end of the reaction. Both FastDigestTM HindIII and CIAP were
inactivated by heat in a single step by incubation at 65oC (water bath) for one hour
prior ligation reaction.
The ligation reaction of the digested and dephosphorylated pMAL - c2x with HindIII
digested Lambda DNA fragments was performed using T4 DNA ligase in 1x T4 DNA
ligase buffer (New England BioLabs). The T4 DNA ligase enzyme catalyses the
formation of phosphodiester bonds between the complementary cohesive 5’ phosphate
and 3’ hydroxyl termini (ibid). Various concentration ratios of the vector and insert
were used and summarised in Table1. Ligation was performed at room temperature
(22oC) for 20minutes and the ligation mix was incubated at 4oC for 24 hours prior to
transformation. In addition, T4 DNA ligase was inactivated in 1:2 and 1:3 ligation
mixtures with 1μl of 0.5M ethylene-diamine-tetra-acetate (EDTA) and the mixture
was purified using Qiagen spin column (section 3.3.). In addition to the method using
Qiagen spin column, the first step in vector purification was by addition 200μl of PB
buffer to the ligation mixture which was applied to the spin column. The first step was
due to having already isolated vector, followed by the remaining steps of the plasmid
purification method.
Table 1: Concentration ratios of vector and insert in ligation mixtures
1:2
pMAL c2x
1:3
1:1
500ng 500ng 500ng
2:1
3:1
1μg
1.5μg
Lambda DNA
1μg
1.5μg
500ng 500ng 500ng
T4 DNA ligase
150U
150U
150U
150U
150U
13
3.2. Transformation
3.2.1. Selective media
Both vectors, pUC18 and pMAL – c2x
contain the lacZ’ gene with MCS. As
already stated, the enzyme product of this
gene enables the host to utilise X-Gal and
produce a blue chromogenic substance.
Therefore, the transformant colonies with
the vector were blue and the transformant
colonies with a cloned lambda fragment
were
white
due
to
the
Figure 2: Transformed E. coli colonies
containing pUC18 vector on a selective
media for a blue / white colour selection
insertional
inactivation of a lacZ’ gene (Figure 2). Selective nutrient agar medium was prepared.
Nutrient agar was sterilised by autoclaving at 121oC for 15 minutes and was kept
molten at 55oC in a water bath. To the liquid nutrient agar (55oC) the following
solutions were added in final volume concentrations as follows: 40μg/ml of X-Gal,
0.1mM of IPTG, 100μg/ml of Ampicillin and 50μg/ml of Oxycillin. Plates were
poured and wrapped in foil to avoid hydrolysis of the light sensitive X-Gal. The plates
were left to set and were kept at 4oC, wrapped in foil, prior to use.
14
3.2.2. Transformation reaction
Competent cells, Escherichia coli NM522 and JM101 were used. The following
method describes how the recombinant DNA molecules were taken up by the
competent cells, a process known as transformation. Cells were pre-treated with CaCl2
and stored in 200μl aliquots at -80oC. Calcium chloride treatment was used as a
chemical method that induces permeability or ‘competence’ of a cell membrane, when
combined with heat shock treatment (ibid). Cells were kept on ice prior to use. In the
study involving pUC18 vector, 5μl of the ligation mix was added to each aliquot
containing competent cells. In the experiment involving pMAL – c2x vector, 10μl of
purified ligation mix or 30μl of the mix without the purification step was added to
each of the aliquots with competent E. coli NM522. The mixtures were kept on ice for
15 minutes, to increase the effectiveness of the transformation (ibid). Cells were heat
shocked at 37oC (water bath) for 2.5 minutes and then incubated at room temperature
(22oC) for 5 minutes. Cells were allowed to replicate by addition of 800μl of sterile
nutrient broth and incubation at 37oC (water bath) for 30 minutes. Various volumes
(50μl, 75μl, 100μl and 200μl) of the transformed mix were spread by ‘spread plate
method’ on the selective agar plates (section 3.2.1.). The plates were incubated at
37oC for 24 hours and then kept at 4oC prior to inoculation of the recombinant
colonies. This method usually yields over 107 of transformed cells per 1μg of
supercoiled plasmid DNA (ibid).
White colonies were subcultured in an antibiotic containing liquid medium to ensure
selection of recombinants containing vector with an insert (satellite colonies would
also appear white). A sterile toothpick was used to transfer a single colony into 5ml
15
aliquot of sterile nutrient broth with 100μg/ml of ampicillin in sterile plastic universal
tube. Bacterial cultures were incubated at 37oC in a shaking incubator for 24 hours to
allow the cells to replicate and therefore “clone” the integrated vector. Subculturing
and incubation yields high density of the bacterial cells and therefore higher amounts
of a vector for harvest.
3.3. Purification of the recombinant plasmids
Plasmids were purified using QIAprep® Spin
Miniprep Kit (Qiagen) and a summary of the
procedure using this kit is illustrated in Figure
3. The procedure is based on the alkaline lysis
method, developed by Birnboim and Dolly in
1979, which exploits the covalently closed
circular form of plasmid DNA that does not
denature under the selective alkaline conditions
(pH
12.0
–
chromosomal
12.5)
that
denature
DNA.
This
rapid
linear
method
therefore allows separation of plasmid DNA
from the high molecular mass chromosomal
DNA.
The overnight E. coli cultures were retrieved
and cells were harvested by centrifugation at
Figure 3: Summary of the plasmid
DNA purification procedures
(Adapted from: QIAprep® Miniprep
Handbook, 2005)
4000rpm for 10 minutes. The supernatant was
16
discarded and the cell pellet was resuspended by pipetting in 250μl of P1 buffer
containing a ribonuclease that breaks down any RNA that could affect the purity of
plasmid DNA (Stadler et al. 2004) and the suspension was transferred to a sterile
1.5ml Eppendorf tube. Cells were then lysed by alkaline lysis with addition of 250μl
of P2 buffer containing sodium hydroxide. The suspension was mixed by inversion.
The lysis stage did not proceed for more than five minutes as it can trigger irreversible
denaturation of a plasmid DNA (Birnboim, 1983). The suspension was then
neutralised with 350μl of high salt concentration N3 buffer containing guanidine
hydrochloride and acetic acid, which was again mixed by inversion. White precipitate
containing cell debris and re-natured insoluble chromosomal DNA in a clear
suspension containing plasmid DNA (ibid) was attained and micro-centrifuged at
13,000rpm for 10minutes. A compact pellet of cell debris was formed and the
supernatant was transferred by pippeting to a QIA prep® spin column. This was
micro-centrifuged for 60 seconds resulting in a plasmid bound to the QIA prep® silica
membrane in high salt concentration and the remaining supernatant in the flow
through, which was discarded (Qiagen, 2005). Nucleases were removed, to avoid
plasmid DNA degradation, with the addition of 500μl of PB buffer containing high
concentration of guanidine hydrochloride and isopropanol (ibid). The spin column
was micro-centrifuged for 60 seconds and the supernatant was removed. Plasmid
DNA was desalted by washing with 750μl of PE buffer containing ethanol, microcentrifuged for 60 seconds and the flow through was discarded. The remaining PE
buffer was removed by micro-centrifugation for an additional 60 seconds. The spin
column was placed in a sterile 1.5ml Eppendorf tube and 50μl of low salt elution
buffer EB was applied on the silica membrane, left to act for 60 seconds and the
17
plasmid was eluted by micro-centrifugation for 60 seconds. Harvested plasmids were
stored at -20oC and kept on ice prior to use.
3.4. Plasmid DNA digestion and analysis by agarose gel electrophoresis
3.4.1. Plasmid DNA digestion with HindIII
Prior to analysis, 1-5μl of the harvested plasmid DNA was digested with 10Units of
FastDigestTM HindIII restriction enzyme in a total reaction volume of 15μl. Overall,
1μl of the harvested plasmid DNA was found to be a sufficient volume containing an
adequate amount of plasmid DNA for the analysis in most cases. Plasmid DNA was
digested for 1 hour at 37oC (water bath). Condensation effects were avoided by microcentrifuging at 13,000rpm for 30 seconds after every 30minutes. Digestion allows
further analysis of plasmid DNA as HindIII cleaves at two sites of the plasmid, which
gains the linearised plasmid DNA and the lambda DNA insert. Digested plasmid was
stained by the addition of 3μl of 6x Loading buffer (Promega) containing 0.03%
Bromophenol blue, 0.4% Orange G and 0.03% Xylene cyanol FF.
3.4.2. Analysis by agarose gel electrophoresis
Samples were loaded into a 0.8% agarose gel (Sigma) containing 0.3μg/ml of
Ethidium bromide (Sigma). Ethidium bromide is a fluorescent dye that binds to
nucleic acids by insertion into the helical structure and by binding to the individual
base pairs using van der Walls forces. The DNA-ethidium bromide complexes can be
visualised under UV light, emiting orange-red fluorescence (Sambrook and Russel
2001b). Along with the digested samples, 1kilobase plus DNA ladder (Invitrogen)
was used as a DNA fragment marker and 500ng was loaded in a ‘marker lane’.
18
HindIII
digested
Lambda
DNA (500ng) and HindIII
digested
were
pUC18
used
as
1
2
3 4
5 6 7 8
9 10 11 12 13 14 15
(500ng)
controls.
Samples
were
electrophoresed at 100V in
the
electrophoresis
tank,
containing the agarose gel
and 1x Tris-borate-EDTA
(TBE) running buffer, for 30
– 90minutes depending on
Figure 4: Analysis by the agarose gel electrophoresis;
pUC18 with cloned lambda DNA fragments: 564bp (lanes:
2-7, 9 and 12), 125bp (lanes: 8 and 11) and 2322bp (lane 10).
*Key:
1kb+ ladder: lanes 1 and 13
Lambda DNA cut with HindIII: lane 14
pUC18 cut with HindIII: lane 15
the size of the gel. Migration
distance was tracked by the migration distance of the loading dyes. Orange G
migrates at 50bp; therefore electrophoresis was completed when the dye migrated to
the end of the gel as the smallest fragment to be detected from the sample was 125bp
(Promega, 2007). Gels were viewed under the UV light transilluminator and a typical
image of the analysed samples is shown in Figure 4.
3.5. Lambda DNA / HindIII restriction fragments
Lambda DNA (Invitrogen) was investigated for a number of HindIII restriction
fragments. In particular, the detection of a presence of an eighth fragment (125bp)
was attempted. In this method various amounts of lambda DNA (500ng, 1μg, 1.5μg,
2μg, 2.5μg and 4μg) were digested with HindIII (10U of HindIII per 1μg of Lambda
DNA) at 37oC water bath for 1 hour. Also, previously analysed pUC18 recombinant
19
plasmids (W49 and W63) with presumed 125bp fragment were digested for
comparison and confirmation of the cloned lambda DNA fragment (20U of HindIII
per 4μl of harvested plasmid). To the digested samples 3μl of 6x Loading dye was
added. Samples were loaded to a 1.2% agarose gel, with smaller porosity then
previously used (0.8% agarose gel in plasmid analysis), to achieve more intact bands
of lower molecular weight (i.e.: 125bp band). Samples were electrophoresed at 100V
for 30minutes. Images of the gel were taken under UV light transilluminator.
20
4. Results
4.1. pUC18
Images of the agarose gel were analysed and in total 121 recombinant plasmids were
analysed, out of which 112 had a lambda DNA insert. The remaining samples only
showed a plasmid even after re-analysis. In addition, data from stage 3 undergraduate
Molecular Biology practicals were collected and additional data from 65 recombinant
pUC18 plasmids was analysed. The percentages of the cloned fragments are shown in
Figure 5. Only four HindIII generated lambda DNA fragments were cloned into the
pUC18: 125bp, 564bp, 2027bp and 2322bp. There were two plasmid samples that had
cloned in two fragments (564bp + 2322bp and 2027bp + 2322bp); however these
samples were excluded from the statistical analysis as these results could have
occurred as an experimental error (section 5). The remaining data (175 plasmids in
total) were graphically expressed (Figure 6) and the frequencies of occurrence of
cloned fragments were statistically analysed by Chi2 goodness of fit test. Of all cloned
lambda fragments, the 564bp occurred most frequently (50.8%), comprising a half of
the total cloned fragments. The second most frequently cloned fragment was 2322bp
comprising more than a quarter of all analysed samples (26.6%). The cloned
fragments did not occur in the equal frequency, which was very highly statistically
significant with Chi2 = 80.06 and Probability value < 0.001 (Table 2). Therefore, the
null hypothesis was rejected as there is less than 0.001% probability that the result
occurred by chance, and the alternative hypothesis was accepted: There was a
difference between the frequencies of the four lambda phage fragments that have been
inserted into the plasmid and taken up by the E. coli competent cells.
21
60
50.8
% of the cloned insert occurance
50
40
30
26.6
20
15.3
10
6.2
0.6
0.6
564bp+2322bp
2027bp+2322bp
0
125bp
564bp
2027bp
2322bp
Fragment size (base pairs)
Figure 5: The percentages of the HindIII cut lambda fragments intercorporated into the pUC18
cloning vector
100
90
90
Number of cloned fragments
80
70
60
50
47
40
30
27
20
11
10
0
125bp
564bp
2027bp
2322bp
Fragment size (base pairs)
Figure 6: Number of Hind III lambda DNA fragments integrated into pUC18 plasmid
22
Table 2: The Chi2 statistical analysis of the cloned lambda DNA fragments in the
pUC18 plasmid vector
Fragment
125bp
564bp
2027bp
2322bp
Observed
27
90
11
47
Expected
43.75
43.75
43.75
43.75
175
175
Total
(O-E)2
280.56
2139.06
1072.56
10.56
O-E
-16.75
46.25
-32.75
3.25
Chi2
(O-E)2 / E
6.41
48.89
24.52
0.24
80.06
P value
H0 =
0.000
reject H0
4.2. pMAL - c2X
Due to the unsuccessful ligation and transformation procedure there was only a small
number of blue colonies (Table 3), with the maximum of 4.65 x 101 CFU / ml,
appearing on the selective agar media containing the transformed pMAL – c2X vector
without the lambda DNA insert.
Table 3: The number of transformed E. coli (CFU / ml) containing pMAL – c2X
without lambda DNA insert (blue).
Ligation mix
pMAL - c2X
Lambda DNA
CFU / ml
1:2
1:3
1:1
2:1
3:1
500ng
500ng
500ng
1μg
1.5μg
1μg
1.5μg
500ng 500ng 500ng
4.65x101 1.75x101
0
5
0
23
4.3. Lambda DNA / HindIII restriction fragments
Images of the lambda DNA
1
2
3
4
5
6
7
8
restriction fragments separated by
agarose gel electrophoresis were
analysed. The presence of the
eighth fragment, of 125bp, was
confirmed. The analysis of the
agarose gel image (Figure 7)
showed a ‘fuzzy’ band at 125bp
when 4μg of HindIII cut total
Figure 7: Image of lambda DNA / HindIII restriction
fragments (lanes 3 – 7*) with upper band 564bp and lower
band 125bp fragment and lambda restriction fragments
(125bp) cloned into pUC18 (lanes 1 and 2)
*Lambda DNA:
Lane 3 = 500ng
Lane 4 = 1μg
Lane 5 = 1.5μg
Lane 6 = 2μg
Lane 7 = 2.5μg
Lane 8 = 4μg
lambda genome DNA was loaded
into the well. Restriction fragments containing below 4μg of lambda DNA were not
visible. Very sharp bands were seen on the analysed samples containing recombinant
pUC18 with the 125bp lambda DNA insert. Both bands of the lambda DNA insert
migrated the same distance as the visible lambda DNA restriction fragment,
confirming the presence of the 125bp fragment.
24
5. Discussion
An in vivo cloning method in the Escherichia coli was used to clone bacteriophage
lambda DNA restriction fragments into the synthetically derived pUC18 plasmid
vector. An alkaline lysis method using QIA prep® spin columns was used to harvest
the plasmid of reasonable purity that is appropriate for routine experimental use such
as digestion by restriction enzyme or sequencing (ibid). However, this method cannot
be applied when plasmids are used for manufacturing pharmaceutical products, for
instance human insulin, where purity of product without contaminants is essential.
There are other methods for plasmid purification that also avoid the use of RNase,
which is usually of animal origin and therefore it has associated concerns of the
effects of contamination (Branovic et al. 2004). This project investigated the
frequencies of the eight lambda DNA / HindIII restriction fragments that integrated
into the pUC18 vector. In theory, all eight restriction fragments should occur at equal
frequencies. However, the differences in the frequencies of lambda restriction
fragments occurrence were proposed by hypothesis due to the relatively small size of
the pUC18 plasmid. In comparison, four out of eight lambda fragments, ranging from
4,361bp – 23,130bp, are considerably larger than the size of pUC18 (2,686bp). This
means, that the ligation and the integration of smaller fragments is more likely due to
their size and conformation of the resulting molecule. Also, lambda DNA is a linear
molecule with 12bp single stranded complementary ends (ibid), which means that
restriction fragments at each end of lambda DNA (23,130bp and 4,261bp fragments)
have only one complementary strand created by HindIII. Hence, only one end of the
described fragments was complementary with HindIII created sticky ends on pUC18.
Although the above two bases can be, due to their complementarity, ligated together
25
with T4 DNA ligase (Becker and Murialdo 1990); it is unlikely that such a large
fragment would be able to intercorporate into a plasmid that is more than ten times
smaller.
During the experimental procedure, nine false positive white colonies carrying a
pUC18 without an insert occurred in total. This could be due to spontaneous mutation
leading to the inactivation of lacZ’ gene due to a small deletion in a plasmid, usually
occurring due to cutting out of the insert during the cell replication, which resulted in
inability to utilise X-Gal (ibid). But also defects in lacZΔ M15 in the E. coli host cell
could lead to the failure of α-complementation with the lacZ’ gene carried by plasmid
without an insert and failure to produce active β-galactosidase (Slilaty and Lebel
1998).
The results from this project have confirmed that all four lambda DNA fragments,
which were smaller than the actual pUC18 plasmid, were cloned and none of the
fragments that were larger than the plasmid were cloned; out of 177 recombinant
plasmids in total. However, there were two instances where there were two lambda
fragments cloned into a single plasmid (564bp + 2322bp and 2027bp + 2322bp). This
may occurred via ligation of the two fragments and then consequent ligation into the
plasmid. Although, it may also occurred as an experimental error, either by picking
two colonies that lay close together during the subculturing procedure, or because a
single colony that was subcultured arose from more than one E. coli cell. Because the
exact nature of the two plasmids was unknown they were exempt from the statistical
analysis. As only half of the fragments were cloned into pUC18, the statistical Chi2
goodness of fit analysis of data was only performed on those fragments to determine
26
whether they occurred at an equal frequency. The Chi2 test results showed a highly
significant difference between the frequencies of occurrence of the cloned lambda
DNA fragments. In other words, the lambda DNA fragments were cloned at
frequencies that were highly variable. Therefore, the proposed hypothesis stating that
there will be a difference between the frequencies of lambda DNA restriction
fragments cloned into pUC18 was accepted and the null hypothesis was rejected.
The comparison between the lambda DNA restriction fragments cloned into pUC18
(2,286bp) and the three times larger vector pMAL – c2x (6,648bp) was not made due
to the experimental difficulties. Encountered problems were experienced during the
ligation and transformation reactions that resulted in only small number of blue
colonies (carrying pMAL – c2x only) and no white recombinant colonies carrying a
vector with a lambda DNA insert. The problems encountered may have occured
through the experimental procedure, but also due to lower transformation rate of the
pMAL – c2x into E. coli NM522 due to its size. Prolonged incubation on ice of the
ligation mix and competent cells during the transformation reaction could have
increased the rate of the transformation (ibid).
Another aspect of this project was an investigation into the actual number of lambda
DNA fragments produced by HindIII restriction endonuclease. This came about due
to a number of literature sources varying in the statement of the number of lambda /
HindIII restriction fragments (ibid). First, the entire bacteriophage lambda genome
sequence was analysed (Gen Bank, 2006) for the number of HindIII restriction sites
(Appendix I). The sequence revealed only six restriction sites, leaving out the 125bp
fragment and instead having a 6,682bp fragment (6,557bp + 125bp). Therefore, a
27
different strain of the bacteriophage lambda was
suspected. The technical details of the lambda DNA,
supplied by Invitrogen, were reviewed. The following
genotype of the lambda DNA was revealed: cIind 1
ts857Sam7 and therefore it was found that not a wildtype but a mutant strain had been used in this project
and by the SoBCHS for stage 3 Molecular Biology
practicals. In fact, when the bacteriophage lambda
DNA was fist sequenced (Sanger et al. 1982), the
researchers had used the same ‘standard strain’, cIind
1 ts857Sam7 that has an extra HindIII restriction site,
therefore seven in total. Gen Bank also states the
variation in the sequence for this strain, which is I
Figure 8: Lambda DNA /
HindIII restriction fragments
and concentrations of each
restriction fragment (ng) per
0.5μg of digested lambda DNA
(Taken from: Fermentas, 2007)
fact a mutation, at position 37,859bp where Cytosine (5’ AAGCTC 3’) is substituted
with Thymine (5’ A↓AGCTT 3’), which results in an extra HindIII restriction site
(Appendix I) and in two restriction fragments that differ from the wild-type strain:
125bp and 6,557bp (Hendrix et al. 1983). However, the presence of the 125bp
fragment was not apparent when using 500ng of lambda DNA cut with HindIII as a
control for the reference of the intercorporated fragment. This is due to a large
molecular weight of the whole genome, which is 48,502bp in total. Therefore, 125bp
fragment only comprises 0.3% of the lambda DNA (Figure 8). In other words, there is
only 1.3ng of 125bp fragment when loading 500ng of HindIII cut lambda DNA
(Fermentas, 2007). The low concentration of the 125bp fragment therefore makes it
difficult to detect this fragment via agarose gel electrophoresis. Even if 4μg of the
digested lambda is analysed for the restriction fragments, the presence of a 125bp
28
fragment was detected as an extremely fuzzy band that is barely visible. However,
when the 125bp fragment is cloned into pUC18 it is easier to detect as the
concentration of this fragment is 15 times higher in comparison to the 2,686bp
plasmid; comprising 4.5% of the total size of the recombinant plasmid. Lambda DNA
is also commonly used as conventional DNA ladder that can be ordered from any
laboratory equipment supplier already cut with a restriction enzyme (i.e.: lambda
DNA digested with HindIII). In fact, most suppliers normally provide a strain with an
eight HindIII restriction fragments (Fermentas, Invitrogen, New England BioLabs,
Promega and Sigma) and not a wild –type lambda DNA.
In summary, the findings from this project revealed that the frequencies of the lambda
restriction fragments cloned into pUC18 varied significantly. The most common
cloned restriction fragment was 564bp, comprising 50.8% of cloned samples in total.
None of the lambda restriction fragments that were larger than the pUC18 plasmid
were cloned. This indicates that size of the restriction fragment in relation to the
plasmid vector plays an important role in the conformation of the molecule or
successful integration into the vector. Further studies could investigate the frequencies
of the lambda DNA restriction fragments in a larger vector such as pMAL – c2x. In
addition, methods to increase the rate of transformation of a larger plasmid into
competent Escherichia coli or using a more suitable strain could be investigated.
29
6. Acknowledgements
I would like to thank to my project surpervisor Mr Howard N Hughes for all his
support and guidiance in this project and also for valuable comments. I wish to thank
to the Manchester Metropolitan University for allowing me to compete this project as
a part of my degree course. I would like to thank to the Molecular biology laboratory
technical staff and Mrs Noshina Shaheen for all her support and for providing a
pUC18 ligation mix. Also, I would like to thank to Professor Joanna Verran for her
helpful comments.
30
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34
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35
Appendix I
36
The Complete Genome of Bacteriophage Lambda
(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=215104&do
pt=GenBank)
Note: Variation in the strain ind1 that has “T”at 37589th base, which replaces
“C” found in the wild-type strain.
ORIGIN
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
1261
1321
1381
1441
1501
1561
1621
1681
1741
1801
1861
1921
1981
2041
2101
2161
2221
2281
2341
2401
2461
2521
2581
2641
2701
2761
2821
2881
2941
3001
3061
3121
3181
3241
3301
gggcggcgac
ttcttcttcg
acaggtgctg
ggaatgaaca
taccattcag
tgaggtgctt
tgagaacgaa
ccagccagga
ggaactgaag
gctgtcgcgg
gcggcgtttt
agccatgaac
cgaacagtca
ggccggagcc
ccgcatacca
tgggcagcga
aaatgctgct
ggttgccgac
gtgatattcc
cgctcaccat
caaaaaacta
atgatgatat
cggtctggcc
agcgtgcagc
gggaggagca
cggatgaccc
aggagctgga
atggcattct
ttcacatctg
tgaaaacgaa
cgtgggaggc
attattcagc
tggaccgcta
accggcagat
ccatcaataa
gggatactgg
tccgggtgat
gtaagcgaaa
agatttataa
acttcccgaa
agcaggtcga
gcaatgaggc
gctggcagct
ccaacaagaa
acaggaagaa
ggcaacagta
gaaaaaatat
tgcaggattt
acatcgctgc
cggtcgtgga
ggcaatgccc
ctgcatcagg
tatctgggca
aaagagtttg
atgatgattc
acctgggata
ctcgcgggtt
tcataactta
aaagcgaggc
atggaagtca
aactggcagg
tatgactctg
aagctgcgcc
actattgagt
aatgccagag
atcgcaggtg
ccggaactgg
aaagcagccg
ggttaacagg
acagaccgcc
ggaagggcgc
ctacatccgt
gggtgtttat
ggatggtgat
gtcgctgctg
gaagcgtttc
ccgtgaaaag
tgaacaggaa
aaagtccatc
cagtgaatcc
gtatcttaaa
ctccagcgtg
ctttactgat
ctggttttcg
gacagcgtac
aggggatacg
gaaaattggc
gcccgttcct
cgaaatgcgc
tattatgggc
aacctatacc
cgggattgac
ccccattaaa
caaaaacggg
ccgcttcaca
taacccggat
aaaatgggtg
actcgactgc
ggatctcagt
aacactggca
cttgccgctg
cagaaagacg
attgcagagc
tatgtatgaa
gcgaatatgc
acccaccgag
gcgcagacga
atcatatcgt
tcggggagga
ccgaggatga
gggaaggtgt
ccagttcgtc
ttcgctattt
atgtttttat
tttttggcct
acaaaaagca
aacagggaat
ccgccgtcat
gggaggttga
acgaacgcca
actccgctga
aaattgccag
aaaaccgaca
cgctggatga
ctgcggcatt
gttgaatggg
tgggaaacac
gaggtgaatg
gcctacttta
gccgagaact
gcgctggccc
actaatgggc
tcggtggatg
ggctctccga
cgtggctcca
ccgcatttta
tttggcgaca
ttttatctct
gcccgttata
tcatccggtg
agcccgttca
ggaaaacgta
gaacgtccgg
gaccgtgtgg
gtatggggat
cgccacgacg
cgccggaatg
ccgaccattg
ggggcatccg
gtttacctta
ctgacgccgg
atttttgatc
gatggcagga
ttcgtttatg
gcgctgctgg
gattacgccc
cccgtgcggc
gacgaagggt
tggaagtgca
aacgcccacc
cggttatcac
tgaaagtgtg
tctggtacgc
cgggtctttt
agaagcccgt
ctgctgctgc
ggccatgcac
gcggcttttc
atgaaaattt
ttaaaatacc
ctgtcgtttc
gctggctgac
gcccgttctg
aaaatggtat
agaactgcgg
tcgacttacg
agtggtggaa
tattctcgac
tgttgatttc
actgataccg
ttgtccgcgc
cggatgctaa
tgccctttca
tggtgaagtc
tagagcataa
ttatgaaaac
cgtggtatgg
gtggcttctg
tggcgggtta
cgttcctggg
cgccaaaagt
tgcgttttca
aagagacgcc
gcgagcataa
tctgcgaaaa
aagagattga
ccacctgggt
aaaccttcgt
atgctgaagt
cttacctgac
gggggccggg
atgaacagac
gtgcagaaat
tgtatgaacg
tctacggaaa
ccgaaatcgg
aaggggatga
tgaccgaagc
aaaaaatact
cgctggcggc
cgagcctgca
gtgccttatc
actgcatgac
ggagtttacg
gaccggcatg
attcccaccc
ggcggtggca
gatgcagccc
aataacggct
ttccggctca
gccttttccc
attgacgttg
gcctttaacg
cggacacagt
tccggtttaa
ctctgaaaag
ctttctctgt
attttcggtg
cgaggcggtg
gccgaaaggg
caggccagcg
cgtgcgcagg
accgcattct
gggctccccc
ctgaaacggg
gggttgctga
cgggcttcgc
ttactatctc
gcgggccatc
tgcccgtgtc
gcagcgcaac
ccacgttgag
caaaaagcac
gtgcctgggc
tgatgaactt
tgacaagcgt
gagaggcacc
tgttgcctgc
gtttggcctc
tgcctgcgtc
gaccgggatc
gccacctgac
gcagattgtc
aaacaccacg
gatggcagag
cgccggtatc
tgaggaaagc
gctgctgcgt
gtcgatatcc
ctcgaaaaaa
gccggtggcc
tacggatacc
accgcttccc
gcagcagctg
gtgggacagc
gctgcgcatc
ggaagaggat
cggagaggat
ctgatgacag
gccacttccg
acacagcgac
ttctggggcc
gcggatttgg
tgttgcccaa
atgccgccaa
gtcatcgccc
gcgaggttga
agcgaaaacg
gtgaactgtt
tccggatggt
ggcgtttccg
aaaggaaacg
ttttgtccgt
cgagtatccg
gcaagggtaa
atgctgaaat
aggcagatct
ccgacgcaca
gtactttcgt
tgtcggtgca
atatcatcaa
gtgaatatat
tcactgttca
ccgaaagaat
atgaatgcga
ggttattcca
acccttatct
ccgactattc
cgggataaca
ggtaaagcgg
gctgcttttg
attgaaggct
tgtcagattg
ccgcattgcg
aaatggacgc
atccgccagc
tggacccgtg
agtgtgacct
aaagactgga
ctcggtgaga
cggaaagagc
gactcccagc
tggctgattg
gtggatgagg
cgtatctgct
catgggctgt
agcatgccac
gcgaaagagc
ggtgccgttc
actgctgaag
aaaaagcgac
agtatttccc
ggtgcagcaa
gaatgacgcg
gtaaacgggt
tgtctgacct
gcaggggacc
ggacggcatg
agggcagttg
ctttacccgt
cgccatccag
aagctggcgc
agcggcatgg
cacgtttacc
cgttcaggcc
cagcccgaag
37
3361
3421
3481
3541
3601
3661
3721
3781
3841
3901
3961
4021
4081
4141
4201
4261
4321
4381
4441
4501
4561
4621
4681
4741
4801
4861
4921
4981
5041
5101
5161
5221
5281
5341
5401
5461
5521
5581
5641
5701
5761
5821
5881
5941
6001
6061
6121
6181
6241
6301
6361
6421
6481
6541
6601
6661
6721
6781
6841
6901
6961
7021
7081
7141
7201
7261
7321
7381
cgcatcagca
aatgacagcg
ccgcagaaat
gtttttgaac
gagcagatga
gcgatgtatg
ctgggcgcga
gcgtattacg
ggtgactcac
cagtcactgc
aattacgccc
tttatggggc
ctggaagagg
caggaagccc
gatggtctga
gagaaagagt
gaaacgatgg
gaatccgggc
cccgcatatt
ggttttcttt
cggcgacagc
cggaccacga
cggcacgctg
cggcattatc
cgatatggac
ccgtgtgcgt
tcagttgctt
catcggcgtc
aatcacgctg
ggatgacgtc
gaaggtgtcg
gtacagcggt
tgcgatcacc
aatgaccaaa
tgacgtggtg
gcagatcacc
ggaggctcac
gaaaacggcc
tgcgctggat
tgatgccgtt
aacctttacc
cggcggattg
taagctggtt
tgctgaccag
tgtgctctgg
ggcaatcagc
tacgggattt
aatttaagtt
cggagaaagt
cgattgtttc
gatatgtcaa
aagatccgca
tgcgtgacga
ttaagggcaa
gcagtgagga
ccacgtatga
atatcatcgt
agaagctgga
gcaaagcggt
agtacgtgga
acactcaggc
aaggcattaa
gtgagttcac
tgtccgtaca
catgacgaaa
tgtcagcctg
gcttgatgac
gaccggacat
acccgaacaa
gtgcggcgct
ggacatggat
ccgtggagga
agatgctcga
ccgccaccat
acagtcagga
ccgcagcgcc
tgaacctgca
tgcggtatat
agatgagcta
ggcgaaaatt
ccatcgttcg
gcagtgcctg
aagaagttca
gcgcaaaacg
agcgccgtgc
tgcgacaatc
gccagcatgg
tgtgcgcttg
ctgactgccc
caggcccgca
gtcagccgga
gcccgtctgc
acgcccggcg
gacataaaac
gccagtgccg
atgatggctc
atttacagcg
cgggagacac
gcatataccg
caggaggcca
gtcatgcgtg
gagactcaat
ccagcgacgg
gcagcggttg
ggacgcgaag
cgccgcattc
cgtctgatgc
aacgatttgc
cattaccagc
agtgcgaaag
gcgtgggatg
accagcacca
ccggaggctg
atcgtttaac
ttttatgtcg
tgatccgctg
ctatctctca
cggtgaggtt
gccgaagcat
gaatctggcg
agagctggcc
atacaccatg
gaataacatc
cccgaccgac
gttcgatccg
tacccgtcgt
gtcctataag
aaacggcgtc
acgcggtctg
cgcctctgcc
catgattcag
actggcgtaa
gatgaactga
acggggacga
acggatgaaa
gaaaatgagg
taccggcgac
gggatattac
accccgtgag
cgggcagact
cacgctgcag
tgagagtgag
gcagcgggaa
ggtccggctg
gacggctcag
cgctgccggg
ctccacggca
cgtcgcatcc
ccgcgtggtg
ggggaactgc
ggaagcggtg
cggtgacgac
agccggtctt
aacagaggag
cctttaatga
caggccagct
aggaggcact
gttatcaggt
cgcgggcgct
aacaggctgc
ggatggtggc
cggtatgggc
cctcccggcg
acagtaatta
gcagccataa
tgcagtcccg
gcctgtccgt
ttgatgccgg
atgcactgga
caacaactgt
agggcgagaa
cggcagaaaa
aacaggcacg
tggccgcagc
agggggcacc
tgaacacacc
cgcagggcaa
cgcctgcaat
gcaccaccga
cgctgacgtt
ccagcgacga
tttacccttc
atgtacacaa
tttctgcgtc
caaattccgg
atccgttccc
gaagtgaatc
gacccggctt
attgctcagg
accggtgaag
acgcagtccg
gatatcgaag
aaaggctggg
ggctctaatt
gggatgtatg
aaaaagaact
cgcacctatg
cgttacccga
tcagcaccgc
tcatggccct
ttgcccgtct
aagaagaact
ctgccggtca
tgggatcagc
agccggaact
gtcagcgagg
ttacccggcg
cgcggtgcaa
aacacgcagc
ctggatacgc
aggctgaccg
ggaggcgcaa
gatacggata
ctgggtgtct
cgggccagtg
cgtcaggcga
acgttacctt
gactggatag
atgctgatag
tatcaggaaa
aaaccgcccg
gagaagagtg
gccgctgatg
tgggatcagc
cgcgacgctg
catgaacggc
gcagccgtac
cagcgatccg
gggggcattt
gcttgccaac
tctggtcacg
cggtgctgcg
ggtggatggc
gatggacgca
gcaggttgtg
actggctgat
tgcacgtaaa
ttcagccact
cgccagcgcg
cagccgcatt
cgtgctggca
accacagagt
ggcaccgctg
agtgtaaggg
cagtgacccg
gaccccgctg
cggtgctgcc
ctacaagtcc
gacgaaaaaa
atcactaaag
ccgcccaact
tctttttccg
gactggtaaa
gtggcggctc
cgcagatgac
accgccgccg
tcgaagagat
ccttcgatcc
gcggcacgga
cctacgcgct
cgctgttccg
ccgagctgga
gcgatgtggc
tcctgccgga
gctgcattca
aaaactgggt
tgatgctgct
tcggggccat
ccgctcgctg
ggcgctccgt
ggacacccct
gcagccggat
gccgtgccgg
acgggtatcc
ggcgcgcctc
atgtgtttta
tgcagagcgc
agtcagcgat
gctggattgg
aagtaccgca
acggctactc
cgtatgagca
cgaacgagtc
gccagatgtt
caaaagcgcg
gctccggtcg
aagccggact
tttttgccca
cctgggcggc
acagcagagc
cttgaacccg
agcctgacgg
gcattatccg
atcgccgtgc
tcggggatga
atggtggacg
gactgcgctg
gacatgaact
cagaccgccc
ctggagaaac
aacccctaca
acccgccaga
ctggataccg
gaacttgtta
tcccgtctct
gcttcgcagg
gcgcagccgg
atggggatcc
gaaacccccg
gcacaggcgc
gctgcaggta
atgtttatga
gctcataccg
atgctggaca
gttggcattc
ggcacgttcc
cggaccgcgt
gccgcctgtg
gctggcggca
tgagagctat
catggcgctg
cacctctgaa
cctgcgtcgc
tcgcatcatc
gcaggcagtt
ggttgaggtg
gtggagcaag
gaacgccagc
ttccttcaaa
gacagcggtg
catcgtcgtg
caacacgatg
ggatgcggac
gaccaccggc
ggctgaccct
tgtttctctg
ggtgaacaac
gtggcagagc
ctcagccggg
accgtgattc
tgtgcagatt
tggctggatg
gttcattcac
cagcgtgatg
cattgtgaag
ggattttatt
tgaaattgcc
cctgatgccg
cgtgtttgag
gctttcccgg
gtgggcgtac
tctgtgctgg
cttcagtttt
tatggccatc
gagtacctac
gcaggtccgt
tgcagcattt
tgcgtaatct
cctatgcgcg
atgcggtgtc
gtgatgatga
tgccggtgtc
ccggttacaa
gcattctgct
acatcatcgc
gcagtgcagg
ggacaggctc
agggtgtgga
gccatcttcc
tgtttgcgca
aggctgcagt
acagcaccga
caggagggcg
ctgacgttac
acgtgaacgc
tcaactgtga
gtatgaccgt
gcagtgacac
acccggcatc
cgagcaaaga
caaccgcgcc
cctccagccg
ttgcggttgc
gttatgagga
ttgccggaac
cggctttttt
aatgagcaga
cccttcacca
tacgtttcgc
tttacgccgg
ctgccggatg
atgcagaaca
tctgccgtgc
gatatgggcc
cgtgacaagt
ggtgtggtga
gccgtcaagg
aaagacctgg
tattccggac
gtgctgggga
gcacagcgcg
gatccggcgc
gatgagttcg
tggaggagtc
tgaaccgtga
tgaaagagga
aaaatgtgct
tggatacgtc
38
7441
7501
7561
7621
7681
7741
7801
7861
7921
7981
8041
8101
8161
8221
8281
8341
8401
8461
8521
8581
8641
8701
8761
8821
8881
8941
9001
9061
9121
9181
9241
9301
9361
9421
9481
9541
9601
9661
9721
9781
9841
9901
9961
10021
10081
10141
10201
10261
10321
10381
10441
10501
10561
10621
10681
10741
10801
10861
10921
10981
11041
11101
11161
11221
11281
11341
11401
11461
tgaactggtc
ggatgaacct
agccgaaatg
ttcgataacc
ggaacgtcag
gatgaccctg
tccctgtttg
ggtgaggaaa
tggcttggac
ccataaaagg
ctggtgccgc
cacaggttgc
aaagggccac
taatcaagct
agcgttcatc
gcgcgtttat
aaaaccgtta
ttaaacaaaa
tgcagcatca
actggatgca
ttttgatgag
cgaagagctg
tcaggtgccg
cgatatcccg
gcgcgacgat
aatgtgagga
ccctgtgggt
ggtcgcgtct
acgacagcta
ctgccggaga
tgctggcgtg
cggtcgatgt
tgatcacccg
gcacggtaac
ggcagagcac
gtgcggtgtc
tgaacggcgt
ctgcggttgc
aaaccgaatc
agcgcattga
accggaagtt
tgtggcataa
ttgagcagga
tgtaccggct
acgccgggcc
cctgaaactg
atccacggag
gctggatatg
ggatatgcat
agatgatgtg
cgggaatgaa
gctgatgaca
atctggtcgt
ggcgtcattt
cgctgagccg
ccgccatgcg
aaagtccgtg
tgatccccat
cgctggcggt
ccgatttcaa
tgctggtcct
cactcagcgc
agagtgtggc
tcgggaagct
acgtgtcggc
gggcattgca
tgaaagagaa
ccatgtggga
acggtcgtgg
gtggcatttg
acagagcgcg
tgttcgatgc
ccaccattac
aaaatatcag
tccggactga
atttctgggt
ggggcgtacc
tcttgagcag
cgcaatggcc
ccgtgagaca
ggtcaaaaat
gggtaatgcg
cctgaaaggt
tcagcaactg
ccccattgat
tattgagcgg
actgaggatg
ctggagaagc
gcggattttc
gacagcgata
gattcagagc
gcactgtcag
gatgcgggct
cgctatgcct
ttataagggg
ggcaaaagtt
tctcgatgat
taccagcttc
gtttaatgaa
gttccgtggc
cacggtgaaa
agcggcaacc
cacgctgacc
tgcggataaa
tgctgcaggc
agaaattacc
atttgaacat
gcatctcgcc
tactgtggaa
ccatccgcag
agtgcttacc
gtctggtatg
cgcagagcct
gcgcgtgaga
tatgccgact
cacttttccg
ccgctggatt
ctgatgcaga
gttatccccg
gtatcagaag
tgatttgagt
ttctggtacg
acaggcgctg
tatgctgcct
gctgatcctg
gttcaggggg
ggcgaccggt
caaaacgctg
gtccagagcc
actggttaag
gcgtttctcc
gaccacagac
ggagcagatt
ggcggcgaac
catgggcacg
tgcggtgctg
cactggtgaa
tgctgccggg
gcctggccag
tgccattgcc
atccggtgag
ctatgccgga
tgaggtgcgg
agatcgggtt
gcctgccgtt
gccgttgaaa
attaaccgcg
aaggtacgcc
ccgcaggcca
cgggttgtcc
ggcggcagcg
aaaaatggcc
gtggtgaaaa
atacggcgtg
gtaataaagc
atgacaccgg
cggcagttgc
cctggcaggc
tggatgcgtg
atttgatcac
tgtggagttc
gtaccaaatc
agcggtgacc
aaagacctga
gaagatgcag
acgctggcgt
ggcgataccc
tgggtcagca
gtcaccaatg
ggcatgaccg
gtggccttcc
acaaaagcca
aaggtcaaca
gtcaccgcca
aacggtgtga
ctgatgaaac
gacgccatca
aagacgcaga
acctggccca
tatgagtttg
gtttctgcgg
tggggcgacc
ggcaccgctt
ggctgacgta
tcagtctgct
aagcggcagg
cttccccgga
ggatcgcagg
ctggatgcgg
gaaagtgatg
gctgcacaga
gcacagttca
ctgcaacagg
cttgccggtg
gcgctggcgt
gtcctttccg
gggcaggcgg
gcgggggtaa
tctgcatccg
ccgacgtcgg
gcgtatgttg
gaggccgcaa
ctggagacct
gatattggtc
gctgcatact
aacggcgttt
aatgcaataa
cgcgccgatg
cagtcaggtg
cagggcgtgc
cagctgcggc
tcgccggatg
aaccgtcgcc
acctcagccg
ttgcttcatc
ggaaactggt
gaatcaaagt
tttcgcgccg
tgcttgtggt
ggtggcatgt
tcccgatggc
aacgtcttcc
gatgaaacat
ggcgacgttt
cgtttatctc
ggagctgcat
gatggagtcc
cagtatggtg
agccgatctg
ctacaatgcc
cttacgcgaa
cgcccggcga
actggactgc
ggatgcccgg
gtgcctataa
gtatcggtaa
tgggacgtcc
tgacgcctgc
agccggaggg
ccgtgtcggt
ttccggttgt
gttaatccgg
ccgtcacgct
ggcaggcaga
gaaccggcgc
tgccgtccat
cggaggcaat
tggtgaataa
gaaagtgttc
cgactggcgt
ttacagtacc
caccgtgctc
gaaccggcgc
gcttgccgga
tgtggcggac
aggagtccgg
ccagatttga
cgaaaaaaac
aagcggggat
ccgacgtggc
gggggcaggt
cgatcaccct
atgcctggta
gcaatcaggc
cagggctgac
gcggtgaggc
gcgtggaggt
ggctgacggc
ctcagttgca
cgaaagggtt
gggcagacag
gtcctgatac
gatgcacttc
cgtgtctctg
cgggaggcgc
aaacgatacg
cggtgatacg
gcgttgaagg
gtggagacac
atggcggaag
gctgaaaggg
tatcagcaaa
cgcgatatcg
aaaggaaagg
taaccggggg
caggcgtcgt
gggtaaccgt
catgcagcgt
ggtgccgctg
gaaagagctg
actgaactcc
tttgatggtc
accggcgctg
atcgaagttt
cggatttatc
gccagcggct
acttatgtca
ggtgaaaggt
tccgctttca
actgaccgct
gaccgggcag
agagcagggg
aatccgcttc
ggcggtgacg
gtcgatggca
cagcacctcg
cgtaaccgac
cagtggtatg
atccggtaat
agagtcagcg
ttctgaactg
acaggcggag
gtttctggtg
gaatgaagcc
ttctcatgct
tgcccctgaa
gacggtgagc
gccatgcttg
cattattttc
agcctgtttt
gaggctgacg
ggtgtccgct
atgacggagg
tatggctgaa
cgagcagatg
agcggcagtc
ttccgtcggg
cacgcagctt
gaaggactcc
gccgatggtg
tcagggcaac
gggactgacg
gtttaaccag
tcagattgcg
ggacaaggtc
gatggctcgc
gcgttccggc
tgatgaccag
gactgcgcgg
cgcgcaggag
acgccacgcg
ccggtgtggc
tgtggctgat
cgggtacatg
tggtgttttt
ctccagcccg
gctgaccatc
ttgtcatctc
ggatgtatgg
acggcggtgc
cagtcggcgt
gccaggctga
gatttgcccg
aaaaaggggc
cgtattcccg
gtggctggga
accacggcgt
ggctatgcgc
gtgcagccgt
gccccgctgt
aatacacggg
tcctgcctgc
cggtgatgag
atgactaccg
ttacctatga
gccgggacca
gacgttgact
gagtcctatg
gggcagaaat
cagcaggcgc
ccgaacggca
gcgaaggaag
gaagatcgca
gtggtgaaag
aagagctttc
accatcaccg
ggtgagtttg
atgttcctga
tcagccctgc
tcagacagca
gcgatgtccc
gttaaacaga
gaaaacgtgg
cagacagagg
tgagttttgc
ccgggatgtc
atgatgttct
tcagcgatcc
aagagcctga
ttggcccgga
atgacgtaat
ccggtaggcg
gccagagtca
gttgaacagt
cagtataaag
gcaggcgggc
ttcggcggga
ggggccacct
tcaaccctgt
gcagatcgta
accagcgagt
tccatcagcc
gctgaagcct
cagttccata
gatgaagccg
acccgccgcc
gcattcaaat
atgctgatta
39
11521
11581
11641
11701
11761
11821
11881
11941
12001
12061
12121
12181
12241
12301
12361
12421
12481
12541
12601
12661
12721
12781
12841
12901
12961
13021
13081
13141
13201
13261
13321
13381
13441
13501
13561
13621
13681
13741
13801
13861
13921
13981
14041
14101
14161
14221
14281
14341
14401
14461
14521
14581
14641
14701
14761
14821
14881
14941
15001
15061
15121
15181
15241
15301
15361
15421
15481
15541
aggcagaggc
ttgttaacga
ttgaagccgc
agagcgatac
ggctgcagac
aagacgggaa
atgaagcgac
aggaagacag
agcatgccgg
gtcagttcgc
ccctgctggc
acaaggttac
agcagcaacg
aggcagaacg
cgctgaataa
ggaactggat
gtatgtcgca
cggcgatgct
tgatgacaga
ccattggcgg
ctgcggcgaa
cagcggggat
gcgtggggaa
cgggcagcat
tggtgattaa
atgacatggc
tgttctccgg
ggcttcggtc
tgccgggctg
ggccacggta
gccgccttat
tatgctgcgt
cggcaggaaa
gaaatcgacc
aaaggtgagc
ggttttgaac
tacggtatgg
cggcgtaagg
gccgatccgg
gcggtgagtg
ggacgtatca
agcggtccgg
tgcagcaaat
ctttccatta
cgcacgcccg
gggaaagata
gccggaagac
ccccggtggt
gccgtggtgg
cgggcggcgc
tctggcgggg
gaatctctat
acagccgggc
ttactgcggc
gtacaccgac
atctgccttt
ggggctgaag
gacggctggt
cagttacatg
gccaagtcag
tttaccgccg
ggcatcctgt
ccgaaagcca
tcctcactgg
cgcgtggggt
caggttgtgg
tttgtcattt
gaagcgaagg
tgcgtataag
tgaagcgcgg
ccgaaagaag
cgaagcgtca
gccgctggag
aatcctgcag
gctgaaaaag
tgctcatgct
agcaaatgag
ggtactggag
gcataaagat
gtatcaggag
ggcaaaacgg
ggaagccacg
cgtcatgtca
ggcaggcctg
ggtaaaaagt
gaccggcagt
aattctgctt
ggctgttggt
attccatttt
tgttcaccgt
tctttaccgg
ggcagacagc
caacgacggc
ccgcaagggt
aggtggacga
ccttctgtaa
aatgccaacc
ctggagtcgt
gagtggcggc
gttgagttca
cactgaatga
tgacagaggt
cggtcacctg
tgaatggcaa
tcaccgggat
tttacgcccg
agcaggaggt
cctcctttgt
tgctggccaa
ctgtcgcgga
gcctgagcgg
acaaactttc
gcgatgtgcg
tttcccctgc
tggctgcagg
ctgccctggc
ctggtctgcc
tttgagcacg
attgagatgc
ctggataatc
gatgtgctgc
gacggcgagc
aaatggcagc
acggggattt
ccatccgggc
atcaggtacg
agactctgcc
gtggcgtatt
gagccaccct
tttctctcgg
gaactccccg
ataacatggt
cacgcgtggt
tgattggtcg
atggagcgtg
acaacctgaa
aaagcagacg
gcgcgttact
gctgagcagc
cggctgaaat
aaatataccg
gcggattaca
ccgaaacagt
gccctgctga
aaaatcagcc
gaggcggcgc
gagacgctgg
cgcctgaacg
gccgccattg
gaacagcgcc
gagcagaaaa
aagtccggct
gcagccacgc
gagcagaact
aagcaggcaa
ggcggcgcat
gcaaccggag
ggtgagtttg
ctgatgcgcg
cggtcgcagg
acgaacgggc
gcccgtgatg
tgaagacctt
gaaaggtgcg
tgaaaacgta
ttctggaaga
agataaaggt
gcgcagagtt
atgcacccgt
cggtggagaa
gcaggggcga
aggcaccagt
ggcggaagat
ttttctggat
gatcagccgc
actgtccacg
cacctgcacc
tgaatatgac
ttgtaagttc
gcagtaaatc
ccagcggagt
gtgaatatct
cagaaatgca
tgagtgaggc
gggggacgat
gtgtgacgga
cggactttca
tggaggcgac
tgtgctgttt
tgctgcacca
gacgcacaca
acaacgattt
actggccaca
gattgccggg
tgatggcgct
ccagattgtc
tgcagcatgg
tgccagtatg
tatacagaca
tgcccagggc
ttctcaggag
ctgatgcaaa
aggaatgggt
gtccacgcag
acatctggaa
gggatgatcg
agactcaaca
ataccgaaga
cccgtcagga
acacgctgat
ccagcgtgaa
cgcttcaggc
agcagcgccg
aacgtcgcca
agtacaaacg
cgctggcgca
atgcgaaaag
tgaaggaaca
agacctgggc
ggagtgagtg
agacctttga
ggcgcagctt
tggtggggat
ccgcgtcagg
gatttacggg
tcttcacgaa
gctatgccac
cgtccgggac
agataggtcc
aaattcagac
ccgctggaaa
ctttggtgat
cagcgtgacg
gcacgggggc
gacctgcgca
tgaacaggtg
gcggagcagt
cgttattttt
cagtatcagc
acgcgcccca
atgcagagtc
gcggtgaact
tggcgcattg
ccgacggaaa
tggacctatc
cagccaacgt
cgcaataacg
ccatgacaca
cgtgcggctt
ccggtgagcc
gggtgagatt
cgaccggcgg
tcataagttc
ctgttacaca
tcgtgaggat
ggggctgtat
tggttcatca
tattcctgaa
ctccctctgg
ggtcgccgca
cagctcccgg
cgggacgtca
gtaattcata
ctgggggctg
ggggcagcca
gtgctcggtg
acggataacg
aatgttctgc
atcagcacgg
atgttttatg
aaaggaagca
ttgctgagtg
tctgcgcaag
tgaaaaggcc
ggacaaaaat
ggcgcagaag
agaactgaac
ggcggcggcg
ggtgtctgcg
agaactccgg
ggatttgtgg
gctgtctgca
ccagctggct
gcaggcggat
ccgggggctg
gtatggcgat
ggctgaagac
ggaagagagc
tggtattgca
cacccgttcc
tgtcgggagt
cggtacagcc
aaccggcggc
ggaggcaacc
cggcggttat
gtttgagcag
ggctgctctg
acagatgcgt
gtgaaacccg
ggctattctc
ctttctgtcc
tggaaatcct
aaatggtcgt
gtgaactgat
cggccagcgt
tctgtaatga
cgtatcccat
cgctgacggt
tggtcggcgg
tcgtcaacgg
agcagtgcag
cggatggcgc
gcggtgacga
ccgatatcac
tcggcaactt
gacagaatca
cgtggtaagc
ggaggctatt
gtggcgctgg
ctgcaggtgc
cgctgtgtgc
ctgttccggg
gactggtggc
caggtgccgt
gtgccgaatc
caactgagca
cgtcaccggg
tcgaccttcg
cgtttcgtca
gcacgtccgg
ttgttcccag
ccgccattgc
ttggggccgg
gtgtggcgca
gtaagcagaa
ctgttctgta
cagacgaagg
tgaaaccgcc
gtaaggggca
tgatcgatgc
gatgattatt
cgtcttgcgc
gcgcagcagc
gcttacgaac
aaggcactga
aaaaaggatt
ggcgatcgtc
acgctggaga
aaggcggaga
caggagaaat
gcacttggcg
aaattcgcac
actgaccggc
aatccgctgg
cagcttcgcg
gccacggaca
cagaatatgg
gtgctgtcca
atcggcagcg
attcaggccg
aaatatgagc
agccggattg
gtcggtacac
aataaccatg
aaggcggtgt
gatggtggcc
gtatggatgt
agcgagcgcc
cccgtgagga
ttctgtggac
cgcgggtcag
gcaggatatc
ggtgctctgg
gcagaacgaa
tcaggggagc
ttctaacctg
aacggtggtc
aaacagttac
cgaactgagc
tgtttttccg
gtgcggttat
gaaggataaa
tggcggcttc
gcgattctgg
acgccggagg
tccgtatgtc
tccacagcca
agagtgattt
cgcatctcac
atgcttatca
gtaacggcca
tgtcagcggc
acgccgcaat
aacgagagag
catggcgcgc
tgtgaaaacg
gaaactgagc
gttaacggcg
agtcgccggg
cggatcattc
tggtatgacc
gatgctggca
cacctatttc
cggggaaatg
ggacggtggt
tgcgggcggt
taccccgcgc
catcagcgaa
40
15601
15661
15721
15781
15841
15901
15961
16021
16081
16141
16201
16261
16321
16381
16441
16501
16561
16621
16681
16741
16801
16861
16921
16981
17041
17101
17161
17221
17281
17341
17401
17461
17521
17581
17641
17701
17761
17821
17881
17941
18001
18061
18121
18181
18241
18301
18361
18421
18481
18541
18601
18661
18721
18781
18841
18901
18961
19021
19081
19141
19201
19261
19321
19381
19441
19501
19561
19621
gggccgattg
ctggacactg
caggagcaga
gaagtgaaat
cgctttacct
tcggaagtcc
atcaccatta
ccgccgcgcc
ctgcagaaca
ccgaacacgg
agccgtaatt
acgcggcaat
gcctggtgtc
gcggcggatg
ccggacggct
cgtaaggcgt
aacgggcaga
cgcagtaatg
aaggaccgcc
gcgacagagc
atggatgcct
aaaacagaac
catgtaccgg
ggtcgtgtgc
ctgccatcct
gtggaggttc
gttgctgaat
tgcgtgagta
ccggaaaaag
gtgaatggtg
ggggaatatc
ctgctccgtc
acgacggaaa
cgggcggtaa
gcaccggcag
ccgcatcttg
attgcggata
atagccgcca
aacaccgttg
ggttacctgg
gaaaaagtcg
aaggatgcca
ggcaaacatt
agccagtttc
acgccgatgt
ctgacggccc
ggaaagctga
ctcagtaatg
atcgtcgggg
gtggactggc
cagatagtgg
acgtattcga
gcgaacgagg
gtgatcctga
tttgccagcg
tgagtgtgtt
aacgatgcgt
tccggcccgt
gggtacattg
cgtgaagtac
cgcgtcgaaa
gcgtggacgt
tgcgtacgcc
taagacggaa
aatgcggcat
cgttgttgat
cgttggggtc
gaaccggtgg
aaggtccggt
aggggaatac
ctccgccgga
atgacacgcc
tcggtgtaca
gcctgctggt
agggcaaaac
cgtttaatat
aaacgctctg
cactggtcgg
atcatctgcg
acagcggtat
tgtgggatat
tggataaatg
ttggcggcac
gggatgtgct
cgctgacgtt
tggtgatgcc
ataatgccgt
ttgttgaaga
ttggctgtac
tgctggaaac
gcgatgttat
tggcggtgaa
ccggtaccgc
agtccgtcac
acagcgtatg
tccgtgagaa
aggccatcgt
tcacgccgcc
aggtgctggc
tgaccgtaac
ccacataccg
atgcgtgggg
caccgtcgag
ccgtttatga
tcagacaggt
gtatcaatat
gcaaatcggc
attttttcaa
agctgacgga
gtgataagtg
atgtcgcggg
tggttgccgc
ttgtggcgca
ccaccattac
ccgctaaaaa
tgacgatagc
acattgtaaa
cgtcaggtac
tgcttccgct
tgtgttatct
cggtacaggt
cgttcacgct
atgtgcaggt
acagaggttc
aatgtgtgta
gcggaaggtg
ccgtcgttgt
cgttatgagc
aagagcagca
tatgtgagcg
atggccggag
atcactcccg
acctcagtgg
attgcttatg
ggttataaat
gcttttttgt
ggatggctta
caacatatcc
gggatttgaa
gatcacccgc
ggcactggtg
tcagatacaa
cacctcgcag
ccggatgcgc
gtcgtcatac
cgtgcaggtg
cgggcgtatt
ctgggacgga
gctgacccat
ggcgctgtat
ggagccgcgc
cagcgatttc
cgtgcaggac
ggatgatggc
tgaggtgaac
tacgcaggcc
cagccggggg
gcagaccgtg
tgaaatctgc
cagccagacc
gctgataagc
cgacggcgtg
ggagctgaag
cgacgacggc
ggataacggg
agcggtgcag
gcgatgggac
agcggacgac
cttcacgcaa
gcagcagggc
gattgagctg
cccgacggta
tgaaaccagc
caaaccgggc
attcgtggag
aggcaagata
ggataacgcc
gaatgccatg
tattggcctc
caatcgtatc
gggcaaccag
cagcggcggc
tgcggatatc
tgaaaactgt
ggcggcgagc
ccgtactgtc
gacgtttcgc
gaaagtactg
gttctcccgt
tacgtccaca
tatggtgatt
gtccgggaac
ttgccgttgc
gacatggtac
cgggcgggga
tgacggacag
cagtgatgac
tgatggccgg
tggctcacag
ggtatatgaa
cgtggagtgc
aaggctccgg
tctgattagc
ggggtgaata
aaaagcgtgc
ggtgtcacgg
tcctccggct
accattacgt
gaaaccacct
cgtaacggtg
tatctggcct
aggatgacgc
actgaaatca
gactcggagc
ctgcaggtgc
acgtttaaac
ccgcgctacg
gtcatcggcc
atcacctgta
tgctcggcga
cgaccgtcgg
gcgccgttcc
tggattgacc
attgcccgtt
caggcacacc
gatttcagcg
gatgatgact
cggacgctga
ctggttgacg
aaggtaaaag
ctgccgacgc
acgtatgcca
gcgcactttg
cacctgaccg
acaccgaagg
ggcagtgagc
ctggcgctgg
gatccggcgt
acgccgggct
cagtttgagt
acgcgttatc
catgattatt
gccgtcggtc
accgaatccc
agcagactgg
tgggctgtca
agcatggagg
gcatttattg
atattcatga
aatcctccgg
agtggcagtg
acgataaacg
gcggcttttc
accgtgaccg
ggaagtaagc
atgaacggtg
attgttgaca
cggcattcgg
aagaaacagg
gggcgtttta
tgtctttgcc
gtttacggtg
taccggtgtg
tgtgggggtg
cggggaggat
accggtttta
tcggtggtcc
agagacgacc
aggtatacag
cagtggcgac
caggtaacac
tggcagtaaa
tgctgaacag
tggtgttccg
ccgagacggt
ctgcaaacat
caaagggtga
gctgggtgac
cggtggtgat
cggacagcac
tcgatgtgaa
agttcggcag
cgtcgaacta
cggcatacag
gcatggggaa
agtactgcga
atgcgtacct
tgcgctgtat
ataagacgtg
gctacagctt
cgaacaacgg
acggtcgtaa
gcgccgggct
tcggcgcaga
atgccggtat
cgctcgaccg
gaagtggcaa
tgagccgtgt
tgcgccagcg
tcaccgccgt
acggcgaaca
cagaagtcac
tggtgaaggg
ggctggtcag
ggaactacag
cggtatcgtt
attttcagat
tctggttctc
ttggtacggc
acttttatat
gggcgagcga
atctcggcaa
aggagttttc
aaattgagca
acacggagga
acccggcaaa
acgacgtgtt
ccttttccct
tgaatgcgaa
gtacgctgag
cgcgccagcg
atgaccatcc
gtactgtcag
cggtgattta
tgccagcggg
cagatattcc
cgctgggcat
ttataaaaca
gcacttgcgg
ggctattttc
agtcatctga
atggcttccc
acgtttcact
caaatcagta
ggcagtacaa
actgccaggg
attaatccgg
tggcgtactg
agtgttatga
gatttcagga
tacgccggtg
ggctggtgag
gctgggtacg
cgaccgtctg
caggaatccg
ggaaaaagac
gggtaacctg
cacagaccag
acagtgctac
ccagcaggtg
taacccgcag
caacaacatg
acgtcttggt
ccagtcagtg
gaccacacag
gccggtatgg
gacctataac
cagcgccctg
ctgggagacg
tgttacgaag
gtggctgatt
agggcttcgc
cagcaccggt
tgaaatcacg
tccggtcagc
tcctgacggt
actgttccgc
gcagcatgtg
gagtggcacg
tgcagacagc
cgtgagtttc
cacggcccgg
gctgacagtc
ccggattgcc
aaccgccacg
ggaaaagcag
gctgtactgg
ccgcagtgtg
tgatgcggaa
ggagctgctg
gaaagagtgg
gaccaaagac
aggcaaactg
cgggaatgaa
cctgaagcgc
gacaccggac
ctccgggacg
ggcggaaaaa
tgaaagcagt
ttttgatcgc
cggcaggaca
tgatggcgcg
tcggggaaac
gccgtatacg
cagcgtggtc
gtgagaggtg
tgacagtcac
aagtgaaacc
aagggattaa
tggggttcgc
atgagagcct
agcaggtcag
tggattaccg
acgaaagtgc
cagcgtccgt
acggattcat
cagcccgccg
gtcctgaaag
41
19681
19741
19801
19861
19921
19981
20041
20101
20161
20221
20281
20341
20401
20461
20521
20581
20641
20701
20761
20821
20881
20941
21001
21061
21121
21181
21241
21301
21361
21421
21481
21541
21601
21661
21721
21781
21841
21901
21961
22021
22081
22141
22201
22261
22321
22381
22441
22501
22561
22621
22681
22741
22801
22861
22921
22981
23041
23101
23161
23221
23281
23341
23401
23461
23521
23581
23641
23701
acggcacagg
ccacggtggt
tggatgtgga
acgccgggac
gtgccatgac
aagaggtggc
ccggcgatgc
cagcacgcgc
ccggcgcaga
agtcctcaaa
ctgcagcgtc
aggccgccac
cgaacgcatc
ccagggcggc
gcgcctctgc
cgaaggcgac
aagcggcggc
tcgcgcttga
acagcacgtc
cgaacagaaa
gctcagggga
agatgttatc
cgggaatgat
gaatgcgaca
tgcggaaaat
aaaaaattcc
ggcaggtgcg
ggggcaggcg
gcttcctgat
gtctcaggaa
tttggggacg
ttacggcacc
aggggccgcg
tcagtatgga
gggtattgct
tacagccgtg
ggttgttatc
cgttaacgct
gaggcttgca
ctgctggccg
ggtctgcctg
gttttcaaca
tatgacgtgg
tttacctggt
gatacggaag
atgcaggtag
acgaaggaag
gttgatacat
ttttgtgata
gtaaagctga
atgactcaat
aaatatggtt
tcttcgtttt
ccaattacct
atgcttgctt
ttggctctgt
gtattgatta
taaccgtcct
gtcgaagtga
attattttat
aattttaaat
atcacatcgt
attctgccat
acccatcgtc
tgtgcaagtc
tcaatgtcat
tctaaatcgc
tcatttttcc
aaaaccggta
ggtgaacacg
gtacggtcag
catcaccgtg
ggaggatgat
gcgtaacgcg
cagtgcatca
cgccagcacg
agcggcatca
aaacgcggcg
acaacaatca
ttcagcacga
atcaagtgcc
aaaaacgtcc
cgcggcagac
agaggctgcg
aatacgtgca
ggatgcggac
tgaaacgctt
agcccactgg
acaaacaata
gacgcgtcac
ccagattttg
ctgacggcgc
gatgccgcca
gttgcagatg
ccgatcccgt
tttgacaaat
atgcgaggct
caggatggaa
aaaaccacat
aaatcgacga
ggtgctcatg
acagcaacca
tatttatcga
agtgccggtg
ggtgctcatg
gcgggtaacg
taatggcatt
gaactaatga
caaacagtac
gtgatgaggc
cttccggcga
tatcgccggg
cagaaaaact
ccagtgagca
aaacctcgtt
caactgcacc
tgccgcagaa
gtattggttt
tgttcatagt
tttcgtcatg
ctctaactat
gaagtctttc
ttctgagcca
cagctgcata
aatcaattgg
ttaaaaaagt
tatttttagg
tgtcatattg
aagttattct
cacccattgg
agattatagc
tttctgattt
tgactaactt
tttcttcaat
cttgtttttc
atttttcaat
cagaactgca
gtgggctcag
tacagtgtca
tatgaagatt
gcccggccgg
tccgtggtgg
gctgctcagg
tccgccggac
gcaaaggcca
gccaccagtg
gccgccacgt
gatgcggtgg
ggtcgtgcag
gagacgaatg
gcaaaaacag
ggaagtgcgg
aaaaattcgg
acaacgagaa
gctgcaacgc
acagtccggc
cccagattgc
ctgacgcact
ctaccaccat
tggcagggct
gcctgactga
ttcttgaata
ggccatcaga
cagcctaccc
ggacaatcaa
ttaagtcgca
cgtcgtttga
ataacacggg
cccacacaag
ttacaggaag
aaacggacag
cacatgcgca
cccattcttt
cggaaaacac
cagaatgagt
atttattggt
cgatattgca
atcgtggcat
cgcgttattt
aggggaatat
gttccggatc
tattgcgccg
gctggaagcc
tgatattgag
acgttgtatg
atttggcgat
gtttacatca
ttttgagtct
tttccatgaa
atctataatt
tagctctgat
acgccaaaaa
atggaattgt
cgtttctgca
cttatctacc
tatcatgcta
cctggcttca
attgtttatt
taaggcatgt
aataatagat
ttttatacca
gtaagatgaa
tatcgtattg
aacattattg
ccattcagct
agaatccgga
tcctgcaggt
cacaaccggg
aggtgctgcg
cacagagtac
tcgcggccct
aggctgcatc
ctgaagcgga
ccggtgcggc
ctgcctccac
cctcaaaaga
cttcctcggc
ccaggtcatc
cggcggcggg
tatcagcatc
caaaacgtgc
aggggatagt
caaaggcggt
actgaccgga
gaacaccgct
gaatacgctg
gactaacgcg
ttccacggcg
actgactcag
ccttggggcc
tatcgttccg
aaaacttgct
ggggaaaccc
cacccacagt
ttacgggacg
ggctcatgct
tggtttaagg
tttatccaca
tcagggcagc
tacagttggt
cagtattggt
cgtcaaaaac
gaacaaccac
gaaggtgacg
ccgccagata
ctcgttgaag
atttctgaac
cagaagtgga
cgggaggcgg
cttcaggatg
tggaagaagt
tggcctgctg
aaataacgtt
tattatcttc
ccgccaattg
gctgttgata
atacattttt
ggcattgtat
atccaaatga
atatatttat
ttatcataaa
agcttggctg
agttttagac
aatgacaatt
tcaaataaag
tgtatgccaa
aataattcgt
gattcagtta
atgtttaaca
ataagagtag
cgagaatttt
ttataccaaa
gaaagccaga
tgaagccggg
tgacggtttt
gacgctgaat
tcgtcttgaa
ggcagacgcg
tgtgactgat
gtcagctcag
aaaaagtgcc
gaaaacgtca
cgcggccacg
ggcagcaaaa
aacggcggca
tgaaacagca
gagtgcgtca
gcagagcaaa
agaagatata
gcagctcagc
taaggtggta
acgccaacag
tttgtactgg
aatgaactgg
cttgcgggta
aaaaataaat
gttggcaggg
ggtgagaatt
tctggctacg
gtcgcgtatc
gccagcggtc
gccagtgcat
aaaacaacag
cacagtctga
atgaacagtt
gttaaaggaa
cacagtcact
attggtgcgc
tcacacggac
attgcattta
ggaccataaa
catatattcc
ttccggctgg
accatcgggg
tcggtccgtt
acggcacagc
aagaaacaaa
ctgcagatct
atcgggtgtt
tccctgttat
ctgcggttag
aggagaataa
cttttaagac
tttctaaagt
gattattatt
gtattggttt
agccataggc
ctgcttgatc
aaattaatgt
tatagtcaac
gctctttaat
tgcttatgga
agtcgaatga
gagagttaca
aatcttttag
aatatgaagg
tactttcatt
cctttgcctc
tagcccaagc
tgtcatatcc
cgtaacagca
cgttacagca
ccaccatcgc
gattttctct
ctgatggtgg
aagaaatcag
gcaactgact
gaagcgtcct
gcagccgcag
gaaacgaatg
aaagcgtcag
tcatcagaaa
gaaaattctg
gcggaacgga
acggcatcca
agtgcggcag
gcttcagctg
agtgcaacca
atggatgaaa
caccaaccgc
ccgcgattgc
ccgcagcgct
aacaaccgaa
taccgtattt
atattctggc
cggcctttcc
tcctgatgca
catcgggtgt
gtgctgtatt
ccggtacgga
gcagtttcga
gcggttcaac
ctggctggag
ccagcacaca
cattgtccgg
accagcatcc
acaccatcac
actatattgt
aatttataat
gcctcatacc
ctttgtggct
taaaaccgtc
accggaaaat
ctgggtgaag
aaaaagcctg
ggaaattgca
gctgaaccgt
ggagtaatcg
ttagtatatt
tggaagttct
tgaacgcatg
cggttttttt
tgaatcaatt
attggagtag
atttgttatt
ttcaaatgtt
ttgaatgtga
taactcttct
atcttcagga
gtaatctttt
tgttggcgaa
gcagttatac
cgtattagcg
taatttcttt
tgtaataaac
gctatacatt
cattaatgga
tataatctgg
42
23761
23821
23881
23941
24001
24061
24121
24181
24241
24301
24361
24421
24481
24541
24601
24661
24721
24781
24841
24901
24961
25021
25081
25141
25201
25261
25321
25381
25441
25501
25561
25621
25681
25741
25801
25861
25921
25981
26041
26101
26161
26221
26281
26341
26401
26461
26521
26581
26641
26701
26761
26821
26881
26941
27001
27061
27121
27181
27241
27301
27361
27421
27481
27541
27601
27661
27721
27781
tttttgtttt
ttcaagcgaa
aaatctttaa
tgcatgctag
aatacaagtt
cacaggaaat
ttggctctgc
aggaacgtgc
ttgctaccga
ctatgactgt
ccttctcggt
accggatttt
gtcgtaattt
ctaggcactg
gtttcagatt
atttccttat
ctgcctccaa
gtttgtcttc
ttccggaaac
agggatgaat
tgcagtcgcg
caataggaag
tttcacctga
ggagattatt
ttgtaggctc
ttctatattg
catttatctg
gaagaggtag
aacggttcga
aagctctgaa
taaaaacacc
gataatattt
ttgcagtact
gtcgcttaat
atcgaataaa
aacaattgca
tttagtgaat
tttagaaatg
gatagcttca
aatgaattct
tccaatataa
aggatcaaat
tgactcgata
tgcaccaatc
atgaatgttc
gaatccggga
atttaacaca
ggtgttaagg
ctgaatagct
gtagtcaatg
tattaatgaa
gatatcgcat
atagtcattc
cccgcctctt
accgcaattt
gttttccatc
tatttagctt
tttataccaa
aggcttctga
ccatttttca
ggcggaaagg
cgtttgacat
gctttgcact
aaattagcgc
ccatctaagt
tgttttttat
tcagcttttt
gaacaggtca
tttgaataat
ataattcagg
gtcttctttc
atgctgatat
gtttgatctt
ttttaatatt
taacacgttg
tgcggctggc
ttttacatat
acgccactgt
gcatgccact
gtaaaaacag
tctatctttc
atacataact
tgcttcaata
aacttttacg
acgatacctg
ctgcctccag
gaaatttgca
cgcttggtgt
gcacgatgga
aaaatgatct
tgaaacaagc
ttcataaagc
aagagggtgt
ttgttctttc
catcatacct
ttttttcatt
ccttctaatc
tcaacggact
tcacgagtta
attgcttctc
actggggaat
gttcgtaaaa
actaatgact
tgtccagagc
ttatcatcgt
aggctgatga
agccagagtt
aagcggagat
aagtattgtg
gcactaaacg
agtctattat
attccattca
gtgctgggca
gcactttttc
cgtgcgaact
acgctttcat
ttaagaaggt
ctttcaccta
taagtgctta
ttttcaccat
caaccatctg
tcaataacac
atttggcggc
taaaaattag
tctgcttcct
cgatatagtc
agaagcgttt
taagtgttaa
tatgcatgct
cactgctatc
ggattgcgag
aagaagacaa
agttgattca
gcaaaatcta
tatactaagt
ctatcagtca
aaatgttact
gtcaaaatat
ccatggtttt
attttagagg
tgcaatgatt
attattatca
ctcataggag
tggtgaactt
tttttgcatg
ccctaggact
gttgccaatg
ccctcctcat
atcatattct
cttttccaat
aattctgact
aaagagtttc
ttagcaatat
ttcgccgggc
tatacccatt
acctcatcta
actaaattaa
atattttttg
atgtcatcgt
ttttctaatt
gtcctgtcgt
tgcaaaaaag
tccgagcatt
gtactttacc
ctatctgacc
gcgataataa
aaacacctaa
ttgaccgtag
gagttgcaat
aagcagagag
tttcgccaac
tcattcgaag
ccacttgaat
gttccatatt
gtctttttct
cgcctagtga
taccttttgc
aaactgaaac
tttcagagaa
aaattgttgt
tagaattaac
tattaaatga
gtccatgaat
tttcaatgtc
tatgtttaaa
aggaaaaaaa
cttcttcttt
gctcatcaaa
ctcgtaggaa
taaactccaa
aacacaggat
tagtattgaa
tttggataac
tattaatgca
caagtactaa
cttccgctcc
gggtgtgggg
ttcttactgg
gctttgtgct
aaatcacctt
tagtgactgc
atttaatata
tggcattata
aaataaaatc
gttcttgcgg
gtatcaatgc
ttagtcataa
tgataaaatt
cttatcagaa
ttcattatgt
atatggtaga
ccgatagtgc
agagaatttg
gctatgtgcc
acctgcctag
ataaaaagta
agatccctct
aattggggaa
gtagctgctg
tttgagtaat
ttaatagctt
attcaacata
gctcacgaaa
ctgcgaaaac
taggcatcac
tctgtcctat
aatatgttct
taacctttgt
aggtaaataa
tggggaagtg
tattaagcat
ttcatctctg
attataattt
gtggtggtat
gttctcaccg
gactttccac
tattgctaca
caaaggtgga
gacatctact
cagatatttc
ctgtggttca
tgaaaagttt
atctactctc
ttttaaacta
tgggtcaggt
aagcgatcga
aaaatattca
tttaccacac
cgtcacctca
aaagtggaaa
ttctgaaaga
ggctaatcga
accatcgctt
catttcaggg
ttgacctaca
gacagtaaga
tgccttattt
catatagtaa
ctctctttta
cttaacgggg
ccactgttat
tatatagtat
taagccgata
tcgctcataa
aagtcgtgaa
ttatgcaggt
tctctggagt
gcgctaatgc
atatgttgtg
ttgatattta
aaaaagcatt
attatttgat
tttggaggaa
agcatttgag
aactctccat
aactgcttaa
accatatagt
attaaaatta
gccgcagaca
gggtgttgaa
taccacctcc
ggagcggaca
gaattggtta
ttcgttcact
gaaaaaatct
gtcattcaaa
aaacgttgcg
cacttcactc
gaaatgatga
aaaactgata
aaaaatgtcc
ttgacctttc
cgaaaattca
atcaccacaa
agcgggtttg
caggttacca
ctgacctgtc
agtaatgaaa
ttcgctataa
ttcattatca
tttagaatgg
ccagaatttg
aatgtctcaa
atgcaggatt
ccattgcgtg
tgcagatgaa
aatcttgtga
tggatattgt
ttacgtctta
tcatcactac
atacaaccaa
ttgctggcag
tgttctttag
aaatatccct
ttgttttctg
ccattccgcc
aaaggtatag
tctgacaatt
gttacccctc
tttggccata
aatttgctga
agttgactga
aaaccaattt
taaaacattg
ttttctactg
cccttaattt
agttactctc
catcgtattg
tcatgttgca
cgccgaacga
gatagccacg
cagacattca
agaaaagaag
cgtagtgggt
gcgacaggtt
tctgttacag
ttttacagta
tatcatttta
gcttatcaat
ttcaattttg
ttgattcaaa
caagtgcgat
tttgataggt
ctgtcaatgt
aaattagtta
gagttgtggc
cgtcgtatgc
tgatttccag
caccgaccat
ttacaaacgt
gcaagttact
tccgataagc
tccgagtttg
tctataatag
gttgaactat
aagtgcttcc
agagctctgt
gcacccggag
ttgtcgatat
tctcccatat
ggataatgtg
aatggacatt
tttttatctc
actactaagg
gagcttaata
ttatttctaa
gttctcgctg
tcgcttttaa
tttcataaga
tcacttcaag
tatccggacg
ttggaacctc
catcgagtaa
cctctggttc
tagtaaataa
cataaaacaa
actcttcata
ttagtttttt
taaatgctga
cattcttgag
gaggagtaaa
ttgggattct
ggttggtgat
cgataaaagc
ttaaatcact
ctggcaaacc
taagtaatga
ctactaaatc
gattaacata
attttttatc
taacatttcc
taacaaagga
caggaatata
tattaaaata
tattacatac
tagttttcca
tggtgcactg
ttagctcttc
gacttcgtag
ctacagttat
tcagctgcgt
ggcacacaaa
tgatgacaaa
gtcactaata
ttatgtagtc
cgtttctcgt
ttgttgcaac
tcccactccc
43
27841
27901
27961
28021
28081
28141
28201
28261
28321
28381
28441
28501
28561
28621
28681
28741
28801
28861
28921
28981
29041
29101
29161
29221
29281
29341
29401
29461
29521
29581
29641
29701
29761
29821
29881
29941
30001
30061
30121
30181
30241
30301
30361
30421
30481
30541
30601
30661
30721
30781
30841
30901
30961
31021
31081
31141
31201
31261
31321
31381
31441
31501
31561
31621
31681
31741
31801
31861
tgcctctgtc
gagcaaactt
gaaaggtagg
accttgatac
cgccaagaat
tatgcaatgc
atccatctac
caacagccag
ttttcaggta
tggcagcgac
tcagtgttga
gcattgccgc
ttattgcttt
ccaggatttt
ttctcgctgt
cttcagtgat
ccctgtagca
catgacttcg
tttaagtcaa
ggtgggaata
cgctggcgtg
gcaagaaaaa
gttacgtctg
ttctccgctt
tgcatttatc
cgcgagtgcg
agggcaagta
tcgcacattg
gtatgccgac
agacaagaca
ctcattgata
gcaatgttta
catctgacac
aagcgttatc
atcatatgca
tgcgtaatga
tccaggtcac
aactggtttc
tccgagataa
agctcaacac
ttgatgtatt
accaattcat
gcttcacgca
gttccagatc
gttcatctat
ggcggcgcat
tgatgtctgc
cgaataataa
cttaaagacg
cagcgttacc
atccggaaac
gaagggcgtt
acagaatatg
tcatctgctt
gtatgaaatt
aggctatgtg
cccctcgttt
cagaaaggcc
ctacccggat
ttcagccagt
catgtacttt
gtcatagttg
ggccatgtaa
ggaggtaaac
gcaggcggta
ggaagtgaat
tttagcgtta
ggacattttc
atcacgatac
atcgcttatc
cggatcccct
tgtgccggat
ctctttgcat
tgttgggatg
gatatcagac
ttccattgca
ttcgtcagcc
atggtttgtt
tctgattaac
aatttctttt
aattttgctc
ttcgtagcga
cagaggcttg
tgcgattcgc
gtaatatcca
ccttcttccc
cctttaccgc
tcctgcattc
cgttccactc
accgccatca
catgtgctat
cgataactct
atctccataa
aggattgtta
tcgtttccac
cagaatgggg
tctatatcta
ccggatctgc
ctcatttata
tgtaaagaaa
tacagactct
ttttacaaaa
gatactcacc
tgtcgatagt
cagtgcagtg
cgtcttcacg
caccttcgta
gcagtttccc
gctggtttct
ggaaaaggtc
gtgcctgaga
ctcctggcaa
cggatcgcca
ttttattgct
catctttcat
aaaaggagcc
ccgtttaaca
gtttcgcggt
tgctgtctgg
ttcctgctga
aagcccgctg
ctgctttcgc
cttcgtctgt
ccatctcgat
attatttatc
gggaaatacc
attatcgtga
gcctcgtcca
tcatcccgct
gcaaagtacc
gctgacttta
gggcatttca
cgcatacttt
tcaaacaggg
acttccggag
atgtcaggcc
tgtgatgcca
tgcttctcat
tcgaaggaaa
gaaagcggtt
ttatcaagtg
gcaattttta
cacttcattt
agtctgagcc
gtaagtcttg
gttatatggc
ttggctgacg
gtggtgatgt
atgtaattta
tcaagccatg
tgtttgtgtc
ctgtctctgc
ttgtttctta
atttctgatc
tgattcgtgg
ccgaacccat
ctgaagtgtc
ggcggcttgg
ctgcgcccat
gttgaatggc
aacaaaaccc
tgtaatattg
cgtactcgtg
atttgtcttc
taccttcatc
acaacattga
aactccttgc
cagtaagata
ggcatcgctg
ccgatctcac
tgcatcctga
tactaacggg
cttgataaca
gacttcgttg
atactcacgc
tactgttagc
ttcccgttca
tgcgtcaaat
gttaatttcg
cttgcacaag
cactcacaac
gtgttgcgct
taatccctga
tgtagctccc
tgccgattgc
gcttcttcag
ctttttttga
ggtgtcattg
ccagaaaaat
caccatcatt
ttctactggt
actcgttctt
tcctcagcca
cagcctcgct
ggatgcgtca
ttttttcgat
caatcacgac
aggcattttt
tggcctcgaa
gttcaaggcc
cgtcgcgata
ttctggcgtc
ccacaccggt
acttctttcc
tggtgtccga
agagtcttgc
gacctgatgc
cgcgacgagt
tttccttcat
cgcctgtttt
cgcataaatc
aacatggtga
atctccttac
cttcagctat
ccgccttgcc
cttcaagtgg
tgagtgtctt
aatgtaacgt
ctgaaaataa
ctaatccaaa
tataaaggtt
ctcttcaaaa
aacagatact
cgacgaactg
aagtacatcg
tgttctttca
atcatccagt
tctccattcc
gccgtagcga
ggtttaatca
ataataattt
attagactta
tacataaaca
taacgcccaa
aatgtatgtc
atactcaacc
tgaagacgac
tctcctttga
acccattgac
tcttgttcga
ggagtcttcc
ctttccagtt
tgctcgttga
gcaatatcct
tccagcagtt
ccccagtcgt
ctcacttcga
tccgacaacc
aatgagtggc
gtaattcttc
actgttggtt
tgatgatttt
caggcttaaa
tacgctacgg
tttcagaatt
aacaagtccc
gcattccgtg
tccagctttt
attggcacaa
aactcaacag
gccgctgtgc
ttgtaacgga
tcgccattgc
gaactccggc
ataatgcagg
tcgcgtcacc
accaccgagc
gttgccgtca
gatgatcggg
gttctcgtac
gcaaacctca
ggagcggggt
cttatgcccg
agacaaactg
ttttcgtgcg
agatgcaatt
tgatattccg
gctttgctcg
accaactcgt
tgattctgct
ctctgatttt
tgcctctcgg
ctcgtctatg
agcatcaggc
ctgcttgatt
aacggaatta
ctcaatgttg
ctctttaccc
agggggtaaa
ggccacctgt
ctcttccatc
tttcaaggct
caaagtctcc
gttcttcaat
ggtcgtagca
attctcctgt
gttcagataa
tctatatgtt
tgcacggtat
taaaccttca
ccttcgtgat
tctttttgct
gtttcagcta
cgatgtttga
gcgaaattca
tgcgaatgcc
ctccaacccc
ttaactgccg
caggatggcg
tagcaatacg
gttttgattt
cgttctcctg
ccagcacaat
catgcattgc
acctctctgt
ctgaacgacc
agatatagcc
tatttctgat
aatacgcttg
gcttttcatg
tgagtcggtg
caaatgtcat
agcctgacgg
atgtcggcaa
gttgtcatac
gtgaaaggga
acctgattcc
aagatgcttt
tttcagtgga
gtagacgaaa
tccccaaata
acgatctcgt
ccttcacgct
cacatgctgt
cggaacttca
ctgcataaac
gattcagtaa
tgttttcccc
gcaagcaggg
tttgctatca
agaagatgtt
cgcaactcgt
cgcataaaat
atggtttctc
agagcatcaa
acataaagat
tgcccggtaa
gcttgataaa
gctgcgcgag
aatgcatcgc
tatccattga
agacccctcc
cctctgctgg
tcactgttga
gcctgtatag
gtccttgggt
tcccggcgct
tactggtcga
cttaaccgga
tcttggacgt
gcaattacac
tcgaatattg
gtcgttgatg
gactcggaag
aataaatccc
ttgtacagag
cagtcatttc
tggaatattt
gtctgcatgg
cagactctaa
aacggtatca
gtacggtcat
gcattttcac
agcgtcagac
gtaatagcga
cagaaactct
aacaacaaga
cttactccca
tgctgtttca
gtcgcggcgt
cgatggtgtt
ctgctctgcc
ttactgataa
aggcgtcttc
tggtggttca
gctgaatcaa
agggtgaatg
ttcatcgttc
tgaatcccat
cgacgttttt
gcaatgctgc
gcataagcac
ctggtttctc
tgcggctaac
aatttgagca
gtgcatacag
tttcggataa
gtgattgcgc
caaaaccaat
caaaactcgc
tcatacgcgg
actgcacctg
tgaaatcccg
catcgggaga
cattcacgcc
aggccagtgc
tgtggaagta
cgttgtgaac
44
31921
31981
32041
32101
32161
32221
32281
32341
32401
32461
32521
32581
32641
32701
32761
32821
32881
32941
33001
33061
33121
33181
33241
33301
33361
33421
33481
33541
33601
33661
33721
33781
33841
33901
33961
34021
34081
34141
34201
34261
34321
34381
34441
34501
34561
34621
34681
34741
34801
34861
34921
34981
35041
35101
35161
35221
35281
35341
35401
35461
35521
35581
35641
35701
35761
35821
35881
35941
ttctgaagcg
agctctcaca
tgctctgcgg
tctgacgatg
tcccatgttt
accggagtga
cgctcggctt
atcatggctt
ctgccttcgc
atcggatgat
aagtccatgc
accaccggaa
ttaaggccgt
cctttaaatg
ccgacacgtt
cctctgaatc
ctgccatgcg
tggccccgtg
ctttttccat
gacgcgccca
tcctgaaata
agggggttag
agcgtcgacg
agtctggata
cgataaggcg
aagggcagcc
tacataacga
cggccttgat
ttattccttc
cgaactcaca
gttgcgccag
cagagtctga
cgatttcaat
acatctgttc
tccaataatc
actcgttgga
ccactatcag
tcgtattgcc
cttaaccatg
ttgattttta
gaggatgctc
cggtagtaaa
ctacagcgag
atccactcgt
tgatcgttat
atgagagaat
tagcaattaa
taagaaaacg
aagagcaatc
caaaacaatt
cccgatggaa
ttgccagcta
ataagaggaa
ccaggcttgc
atctgtttgt
cgtgatttcg
ccaaccaaca
cggcgtgttt
ttgtagtcct
cttacggcca
ccttcttcag
atgtgctcag
cgcagatggt
ggtaggtgag
atacaattgg
cgggttattc
tgaacgatgc
aaagaagcaa
gtgatgacgc
tcgatcccgg
ctttctgttt
cacgaatgtc
tatccagggc
tgtcgcgttc
catccttgtc
tatgacgtaa
gagttttgaa
tacggtcctt
catcaaactg
cgatgccatt
actggttggc
ccgtctggcg
cagccagctt
aatatcaacc
ctcctgaaac
gcgttgcaaa
gtcgtctgcc
gctctgagcc
gctgtgaaaa
tgaatgcttt
gcttcacgaa
gccataagtg
tttccatccg
agcaacaggc
aaaacgcctc
agtcatatca
gctaccatcc
cacaacacca
catgattaat
ccagaaatta
ttctattgat
atatcctcac
agaacaagtc
atgaatacac
gcagctttgt
catttatcga
cattccgatt
tcaatagtcg
ttcgaactct
gattgtgcct
aaatcggata
tattctcgga
ctgggttgga
cggtattcct
tgtgcatcga
cattaagatg
tatggttttg
cccatacatt
tggaaagcat
tgatccatat
tcgattttcc
tgtaccatgt
ggtattcagc
gtttgcgatt
ggggatttgc
gtgcatccat
gaacgaaaac
atgcttcgtt
ggcttaattt
tatcaccgcc
tatctgtatg
agatctgaat
ttatgtgttt
ttgttctctg
agaggcaatg
taacccgcag
cgagccgtaa
tacgctgcag
caggaatcca
gcggcgaaat
gatcagcaga
cggctgacgt
atagatacca
catccgtttg
tggttcgcgg
gcggtaaatc
ctggttttca
ctgcttatca
aacgatcagt
aagagtggtg
cccagccagc
tggtggtgag
tcaacatcgt
tgatcgatgc
agttctgcct
tcaagacgat
tatcgcccgc
tgcttgatct
acatcttttc
tttgatccat
tcacgtaatt
caccctgcaa
gagtgaagcg
tctgaatcaa
attggaggcc
tatgcattta
acagcattta
ttaatctggt
gctttccagt
agataaaaaa
ggctcctgtt
agtgcagtgt
tgttctgttt
catatttccc
gcagcttgca
tagtcatacg
tcaaattctt
gtcttttaac
aactattaca
cgagtgttca
cttctgcttt
catgtgtggc
ttatcagcta
caaaacgata
tgcgcagccc
agtgagttga
atattattcc
aatattgaga
cttaattttc
gcgctgattc
cagcactgta
cagcgagaga
tgctttccat
ctggattctc
cccccgcgat
tcgtatcaca
ttaagagcgt
agtggtattt
ttttttatat
tgctatgttt
tgggggcgat
gtcaaattat
ccgatggcga
aaaaacaaag
tttgtgccac
gataatgtcc
agagctttta
atctgggaac
gtgttaatct
tctgcagtgt
gcaaatccga
ggatgcgact
cggcattcat
cggcatgtac
ttgatgatgc
ggaaaggcgt
aatgcgatga
atcagttcct
gttgcgagtg
caatggtttc
catcaaacgc
atagcgattc
ctttctcttc
cctgaatgta
gaaatgccgg
cagtttcagt
atcgccaata
tctttgggac
tacgggtgat
tggcatattg
ttattggtat
atattcctga
atccttcctg
agtcgcttga
atacagagcc
gaagtttttc
cgtaatcaat
tcgccctcac
tagttacgag
ttattctgtt
accaagttct
atcttccatt
tccattgcat
gatagtcctg
cttccatata
cacatcaggc
acccctacag
gtaatgaacc
taagcccaga
atgttttcgt
ttgccagcgc
aagtgcgatc
ttaatgaagg
ttgagcttgg
ctattgagga
taaagccaag
tggcgtccac
ttgcgctcaa
aggtctatcg
atagggcggt
tgagcctgtt
ctgtcagtta
tggcacattg
caccccaaag
caccttcatg
atgtcaacac
gaatttattt
agtgagttgt
cgtgaggcaa
atagttggaa
tagtgggtat
ctccaagctc
gcatcatccc
ggtgtcatgc
ctgcttcggc
agagcggcaa
cctgcatggt
atgcagtatt
aggccagacg
gccacggccc
ccatccattc
aggattcatt
gggaccagcc
aaatttcttt
actgcgcatc
gtgggtcgac
cagtactcat
aaccatgtac
acgggtaatg
aaacaggtgc
acgggcgagc
ataagcgttc
gctgattagg
attaatatcc
aaagtggcga
tcctggctga
tcgttcaagt
catggtgtgc
gcggtaaaac
tgtatcgata
accatttcca
aattgctata
gtgtttattg
ctctgtcatt
gatgtatttt
actggagggc
cgacattgct
atttatgcca
ctggcaatca
acaggaaaca
cgcttgaatt
gtattgttcc
tcaccttaaa
tcggtggttc
tttgatgagt
tctggagaga
taactggcct
ctttgctctt
cagatataag
agtaattcaa
caggaagtat
tgtgttgaac
tatttactgg
gccaatatct
tgcatgttat
tacgttgcag
gatttagtgc
taactggttt
tctctgcgcg
gctttggtgg
gcagctaatc
ccttctgctt
gtggtcagtg
cgccagagat
tttgcagggg
atctatttat
agaaaacccg
aacaaggatg
catgtagccg
aacaaaacta
cctgttcgac
tgccaccttc
ctgtgtcagt
taagtcgtca
ttcatcgtta
ttcgacaatg
ggcacactga
cgtgatttct
ggtaacgcag
gtcctgctca
atcaacgccc
cgtccacgga
gctggcatca
agaatccatg
tcgttttata
cggatgtgtt
gattttttgc
tggggcaggc
tgctggtagt
atggctgaac
aaaacaggaa
attttttata
tagtgaattt
ttaagtatgt
aaagattcgg
tccttattta
cgcactcagg
tcggtaattc
tcattccagt
agcagagcat
agtcggtatt
acgtcatggt
ttgatgtttg
aaagaagatt
ccgtgtattc
aaaataaagg
ttgccgtcgt
tttcttcagg
gtccacacca
atcacatcct
tagtggattg
tcgtgtaccc
atagaaatgg
accatgtata
gaatatgtta
gcattttcgc
cgatttaagc
aaccttacag
gtggttacat
aaaacttttt
actgaattag
aagtaactag
gccgcgttcg
gttgctttca
gctttctact
tgcgcttacc
acgttcgcgg
tgtgtggcag
cggaatcgca
tgaatgctgc
cgtcctgctg
aatttatcac
ggcattgttt
ttttcaataa
gcgctgaggc
catatatgaa
cttatgctgg
agggcataga
45
36001
36061
36121
36181
36241
36301
36361
36421
36481
36541
36601
36661
36721
36781
36841
36901
36961
37021
37081
37141
37201
37261
37321
37381
37441
37501
37561
37621
37681
37741
37801
37861
37921
37981
38041
38101
38161
38221
38281
38341
38401
38461
38521
38581
38641
38701
38761
38821
38881
38941
39001
39061
39121
39181
39241
39301
39361
39421
39481
39541
39601
39661
39721
39781
39841
39901
39961
40021
caataactac
ttccgattag
acaatgtcgc
ggaaaaggag
gcatgattct
ttttaaaaca
ctatctcagc
aatgttctgt
gaggtgacgg
gcttaatgac
cctttaatat
tgaaatgcat
cgcgctctcc
tggtcggccc
atgggaatcc
tagagcgatt
ttccatcgat
ccagcagaga
tgctgcggta
tctgagggga
atgcgccgac
ataactttcc
agtggttgta
cccccaagtc
attaacattc
tcaacctcaa
gcatcacctt
aaaacagggt
tcgtagattt
gcaagcaatg
cctgactgcc
ttcttttttt
ggtttctttt
gtgcgtgttg
atggaacaac
aaagatctcg
tttttaacta
aacaaaaaaa
atcaaattaa
tctaaggaaa
gagtgcgttg
cgttgataag
gcttgctgtt
tgctgcgatt
gatggagttc
aaaatactca
gatgatggtt
accaagcgac
ccaatggaca
tgcaatgaag
tttggaccaa
aaaacgaggg
acaaaagaca
tcctctgacc
ggcagcaagt
aagactatcg
ctgatgcgtg
tgccaggaca
acccaactcg
ctcgacctga
agatggttaa
acgacgaaaa
tactggcaac
gtcgccagtg
caggaatgcg
ttgcatggtg
ttgatatggt
cgtggaaatc
cgatgtcata
aaacgtcaag
cccaagacca
atagcaaatg
gttccgcata
ttccagtata
attggtgacc
taaaatatct
gttaaaaata
tatatccaat
atcgccaaat
atgcataaca
actgcttaat
gaaaacagtt
caatgattcg
tatcttctga
tttatgaata
attaaggaaa
agtcgcataa
gtgaaaattc
cagaacacct
ccacaacgga
aaaacacctg
tggctatgca
cgtcaggaaa
gccagaatgc
tggtaaaggt
actcatactc
ctctggcgat
cggcgttata
ccatccccat
cataaattgc
ttgtgctcat
actattttac
gcataaccct
gcgtatatca
taaacgctga
caacagcata
accacaccta
tacttacata
cttaacaaaa
tcgcagatca
cttgaatggg
ctcaccaata
tgaggtcatt
acttcggcag
acgccagact
agtttaaagt
gaatcaccga
ccaagttaga
ataaaaacat
ataaaacatc
ctattacaaa
agccagaaaa
gggggacagc
caccatcagc
aacgtgacgg
acttctggtc
aaatcaaccg
caaacacaga
ctttgaccgt
gccgcaggta
tttcccggcg
ggttctggct
cgtagcccgt
ccgggaagaa
ttacgagtat
aaacgcgcac
tacccatact
gcagcaatca
tctctatgag
cttacgataa
attactcctg
tcacttttca
ttgttcagag
ccggcctcat
atatccttgg
gagtcaaaaa
acaggtagct
tcgtctttgg
gacattcctt
ctggcaaaac
tcatctgcga
accagactct
tacaaataat
acagacaggt
aaaccattct
ccctaattcg
tgccgatcag
acaactctca
accgctatcc
gaaatcacct
gcttggcttg
agaatcactg
tctaagctca
acttctaagt
tgaagggcta
agcatttaat
cttgtctgcg
tttaaggcga
acgttaaatc
ctctggcggt
gaaagattat
aagcgcgatc
tggaagcgtt
aataaccccg
tggtgtatgc
tggttcgtgc
tcgcaatgct
gcaggtggaa
gggtcgttga
aaaaacgccc
actggatcta
aggtaacttt
atcaaatatg
gctgcttgcc
ttctcaactt
actcgtcaga
ctcagaatgg
cctcaaattg
agaaaaaaga
cgacctttct
agaagacctg
cagaaaaccg
acgtaaccac
cggtaacgtg
taacaagcaa
ctggatttac
gagcagatgc
cagcaggtag
agcctggcta
tttcgggaaa
cggcagaatc
gcatccgtta
tgccggaagc
tactggctgg
ctctaatctt
ggattgcaat
ctgaaaaaga
cgtaaggaat
ataattaatc
ttcttgcgta
gcgctgagag
cttttgcccg
caaccttttt
gctccccttc
tggcttctac
tggttcccct
tcccgattaa
caatggtgtc
ggctgttctt
tgtcatttgt
tggagccaac
ttattgagcg
tcataattca
atgaagattc
ccaaacgtct
ttgcatggga
ctgatcagtt
ggctcaacag
gagcctgttg
gcttttttgg
ggtgagaaca
gacggctgca
aattcttcaa
gcattgatgc
acagattcct
cgtgcgtcct
tatcaccgca
gataatggtt
gcaatgcgct
aacaaggcca
tatgcggaag
ctcttacaca
atttatttgc
aaacaaacgc
tggaactgag
gagggactgg
cgacgacatg
ggcggcaacc
tcaacaggag
gccggacagg
ctgcttgagg
attctgcgta
agcgagatta
atgaatatta
tgcatccctc
ggggattgct
aaagattatt
gtggtgaaac
accgccgcag
aattttgctg
cgcgacatgt
ctgagcccgg
caggcaggcg
ggggtggatc
gtcggatcgc
cgcagatcat
accgtgacca
acgggatcac
gaccatttct
ccgccggact
gaggcctgta
ttaccaacct
ggccagtcgg
catggttcct
aacaccagga
tattactatg
cttaactttg
gcaatatgcc
atggcctttt
caggctaatg
tatatccctt
aatatctgtt
cttcaccgtt
catcagtggc
aaaatctgtc
gccttcaaca
aatatcttca
tttggtaaag
ctgcaggtga
cttatctttc
atccatttac
ttgctcaatt
cttcaggcca
tcattgggta
tcttgaaggt
cctgctcagg
gtgcggtcat
ttgtgcttac
tccctgcctg
tactaaccgc
cgctaacttt
cattaaataa
gggataagcc
caagctgctc
agggataaat
gcatgtacta
ttgggcaaac
ttcatgcagg
aggtaaagcc
ttccagccct
atacattcaa
aacgaggctc
aagacagcgg
attccaaagt
gctcgattgg
gagcgttctg
tcattatgac
agcgtaatgt
cttattcggg
aaacctatgg
caaagttacc
tcaagcagca
aaaacgaggg
atccctcaaa
cgtcagagaa
cggatgctgc
agtggatgtt
ggtgggctaa
gtgtgctgtt
ccaaactccg
tgacagccag
tatgaaaaac
caacaacatg
caacggtgtg
gaacgaagtg
cacgatggaa
gccatcaccc
gccaaacgtc
tccggatgcg
gtatcagaac
cgcgttctgc
gcatatgatg
atgtagtggc
taaacaccag
cccacctgcc
atctcttcag
tctgatagat
tctgaaaatt
ttaaattttg
gcccctaaga
gttcggccga
tctatctgaa
agatcggatg
aacaaaaaag
actgaagctt
agaaaagttt
tgattatcag
cctttatttt
tatgttatgt
gttatcagct
ctgactagcg
ctgtgggttt
aaactcatca
gtcaacgaga
ggaattacct
ccatctctcc
aacatgagaa
ttcatacatc
gagaattttt
agcaccaacg
aagttcattt
ttgtgttaat
atctaacacc
aggaggttgt
caagacagct
ccgaaagatt
cttcccgagt
gaaaaagggc
tcaattgtta
tacgaatcga
aagctgtggg
tctcaatgct
cgcgacaagt
aacaaatcca
aaatacagca
ggcagatctc
cgcagatctg
gtggaataaa
tgtcaaacgg
aggcggcatg
aaaatcccct
acagggggac
ttctggcgaa
aattcagagc
tgacatggtg
cgatatccgc
ccgctgggca
cgataagtgg
caaaccaaaa
atcgccgcac
ccggaacagt
ttcagccagt
aacgaaatcc
caggttaacg
gggcagtttg
agcgagctgg
gagtcttatc
atgcgggcca
46
40081
40141
40201
40261
40321
40381
40441
40501
40561
40621
40681
40741
40801
40861
40921
40981
41041
41101
41161
41221
41281
41341
41401
41461
41521
41581
41641
41701
41761
41821
41881
41941
42001
42061
42121
42181
42241
42301
42361
42421
42481
42541
42601
42661
42721
42781
42841
42901
42961
43021
43081
43141
43201
43261
43321
43381
43441
43501
43561
43621
43681
43741
43801
43861
43921
43981
44041
44101
atgcgcttac
cgagaattaa
gtagacctct
gactgaaagg
taatagcttc
tcaggccgcg
aacggtgtat
ggtttttaag
taaaagatga
tttatttggg
atcagcaaaa
tagtaaccat
taggtgacgt
agtgtgtgtt
gctttgtggt
tagagcttat
tggctctgga
ctccggtggc
taaagacgtg
tgtcagggaa
cccggagctt
aatgcctgtt
cggcgcgttc
tttcgggcga
aaagccaaag
cctcgcatgg
catattctgc
attgcagcgt
aacgccaaag
ttcagaaaat
cggctcttgt
ggaagctgca
ccaaatcaac
ttaccactac
taatctgcga
tcccaaaaga
ggctaagacg
aagcgtcgag
tcacgtgtgt
ggaagaagat
ggtttcaccc
cactcgaacg
gacgagagga
gcagttactg
gcgacttacc
accggacaac
gcgtggtgtg
gccgcatcgg
ctatcgaaga
atagcagaag
agcatacgga
aaaatacgtt
tcatcgcgga
gaaatatttg
atacgattgg
gtggtgcaga
gtggaaacca
ggctgcttaa
aagctcttgc
aaaaatatgt
ttgatcatca
tgaaagaaat
tcaagtttgc
tgattcaggt
aaaaagcccg
tgatgtgatg
ctgcggtaag
atttgcacac
tgatgcggaa
ccgtggtgag
aaatcgtgca
agcaagtgta
tgtgcgccgg
gctaaaatgg
taccggtttg
gagtgtcgcc
ccatctacat
ggagagggaa
cgctattcac
tcaggaacgc
ctctcgtcag
taccgcagca
aataggccag
acaggcattc
gtggaaagcg
aggacgtcag
cattacgttt
gttgtgaagt
ggacagccaa
ctgaagccat
tgcactgaca
gggaattaca
cctggaatca
tggaagcaac
attaaaaaat
gtttttaatg
gagattatgt
gattatcaag
tctgagtcat
tgatgcgatg
cttactggaa
cgcaggaaaa
aaactgcaaa
atctgacgta
tcgtgcgagg
cgagctttaa
gagcactgct
gatggctaaa
tgcattcgct
acgaagtaaa
gcagaaacag
gattaaacaa
atgtatctcg
tgctgcggca
caaccagcac
gcaggaagca
gtgcaaggcg
tgaggccgca
aatcagacag
gatgataaag
tggagtgaaa
ggtagttggc
attcgacaac
gaacgttgaa
tgagcaaatg
tggcggtggc
ccataaagca
tatctgccac
gcaggtaatc
caaaggcgcg
caaccaaatg
acagggagaa
atgatgagcg
gctgctatgg
cacgaactca
aaggtatcgg
ttacgccgta
gcgatccctg
caggctctgg
tgacgggcaa
acgttgccgc
cacgggcagg
ctaccaggga
agagtgccgc
tactgagcta
gtcatgaaaa
gcagtacagc
aaccgcagct
gttgaatggc
ttaaagcagc
tcaaccagca
ggtacagagc
agatggggag
catatttgct
tcatggatac
tctgggatat
atggttatac
ttatcgatat
gattaaaact
ccacgtggat
gatatcttgc
aaccattcga
caacgcaaaa
aggtcatcac
accgaggaag
ccctgtatca
gcgaaatatt
ttatcggtgc
tcgatggtgt
ggaggacgtg
taccttccaa
aaaaccttca
aaaacaaggt
cgtgcgctaa
gcgcagaact
ccagcgcgaa
aatcagtggt
gaacgcgaaa
aaagataaac
gcccaacaag
tgcggaacgc
cctcaactcc
aaaagcggaa
gtagacgaaa
atcaaggcag
tgacgttctc
aagtagcacg
acgggaaaat
gagatgcgct
gatctgcacg
aaaaaagacc
tgcctggaat
atgattgatg
tggttcttta
gatgaacttc
gccgattatc
tggaaccgcg
gacacgttca
tatatcgata
ggcgcatgag
actcaccacg
ggatggcgca
gccagaacga
ggaaataccg
aggccgcaga
aaccagtaaa
cgaagatcgc
agaggcaatt
gctaacaggc
tcttctggtt
agaacgggaa
gatgaaacgg
ataacaggcc
aactaacctt
aaatccttcc
tagaccaaaa
atggtcgctg
aggatgttgt
ggatgcgtgt
gtggcgttaa
acagggctgc
ctggctaatg
aggttgtgaa
accgctcacc
ggtatgggaa
ggtaaagaaa
cgttcccttc
tggcatcaga
tgaactgtca
tttgcaaata
aatcggactt
gggatcccat
aatgtcgctg
ggacatggta
tggagggcag
ggtgaatgca
ctccggtgtg
tggcgagaca
cgaaacgcac
actacacggc
gattgaccaa
ctgcggtcag
gatgagcgat
gacgatgtaa
ggtgctctcc
aagcggaaaa
ttaagattcg
ccgtaaacgc
tcacgtctgc
gatttaatga
atctcgttcc
tcgaatcaaa
agtaccaaca
agtaaaaacc
cagactgaaa
gcacgccatc
attacgaaaa
gatgctacac
tgcttatctc
taatcacatt
gcttatcaga
atctcgatta
cgttaatcat
cctttgacga
aacgaatcag
tctttggtca
ccggcgcagt
actcgaaagc
ggccacggct
atcacaagcc
caaacaaaag
tggtgtggca
tgagcttgtc
acaacttcct
agaaatcaaa
attcattacc
gcaacagtaa
atcgaaggta
ggaaagatga
gtattggcgg
tgctggtaat
tgaaattcga
agacccaacc
caggaagcta
gctggatgca
tcctaacctt
aggcgaattt
gtggtcagac
atgataaatg
gagcaaaagc
catccaatga
gtattgcagg
ccaaaggata
tatggcactc
accaaatact
gctgatgaac
gactttgaga
ccggaacatc
gcctgcaaag
gtgcgtgacg
gacggtatcg
cgagctaaaa
cttgatttcg
aagaagataa
aaagaacacc
gcgacgaagt
cagaaataaa
tcacctgtgg
aatcgaagtt
aagctgcatg
ccgaatagct
aaacgatgaa
agagtgtgga
agcagcagag
aaaactcgcc
cttcatcaga
tcagtgggat
acgcaatatt
gtatcgcgtc
ccataaccgc
gaaactcaaa
attccagaca
tgtagtcgcg
gtcaacgacg
aattgatggc
gaacctgatg
ggtgggcgat
cccctggttc
gcgtggaaac
cgacaaagaa
cgaactggtg
atacgagttt
caactcacaa
tacgccagca
gttctgcgga
gtagctaaat
tctgactctc
ggattcggta
gctatcaact
aagcttgaag
catatgactg
gtcatgggcg
gctaagttcg
tggggacgca
ccagcataaa
aggtctggcg
gcacgaacct
tatatggagt
cgcaggcctt
tctccagcac
aaaccaatcg
tgggcctgct
gaaagctgga
gccgggaatg
gcggagctat
gaagcgagac
tcgttagttt
gacgggcagg
catatcggtt
ttgatatcaa
ttcagacgcg
catacgtcgg
gtgatgacca
cgaagcggct
aggaagatat
tcggtaactg
atgaggaggg
gacatcggga
cgaaaatgta
gattcgatac
acttcgggag
ccgcttccga
aacaggggtg
atcaccgaca
cccaagccaa
gatatccggt
acgaacaaga
tgctggaagt
cgatgcacga
tgccgggaat
accaagatag
aagaaacgac
ttaaagcccc
gaaagagacc
gccggacatt
cacaagcaat
gaactgatta
catcgctgga
gacctgcgaa
tgctcgttga
gtacggtcag
ttctcatggt
agcaaatacc
aacaaactgg
ttggttgatc
agagctgtac
gttaatcact
attctggcta
agcaaagata
ggaaagccag
aacgggatcg
gtgaaaccac
aacctaacat
ttcattcgcc
tttccggtac
tggctgcatt
atctgatgca
gaaatactaa
47
44161
44221
44281
44341
44401
44461
44521
44581
44641
44701
44761
44821
44881
44941
45001
45061
45121
45181
45241
45301
45361
45421
45481
45541
45601
45661
45721
45781
45841
45901
45961
46021
46081
46141
46201
46261
46321
46381
46441
46501
46561
46621
46681
46741
46801
46861
46921
46981
47041
47101
47161
47221
47281
47341
47401
47461
47521
47581
47641
47701
47761
47821
47881
47941
48001
48061
48121
48181
ggcaaaggta
gacgccgggg
aacagagctg
ttcaaggatg
ccaacccacc
caaagaagag
ccacggatgg
tgactcaacg
ctaccgattc
cagagaggtc
aacggtttcg
aagagtcggc
ggaagcagaa
cccaaactga
cttttacaca
gttttgcccg
tgttctatca
aagacatgaa
aaggcatcgg
gtgcgtttac
gtgaccttct
tcggctacat
ccggagtaga
tggtcggagg
gtaggcggag
ccaaaactca
taccgcaagc
cagcagatta
atcgaccgtt
cataaggctg
gatgtatgag
tgtcatgggc
aaaatgccag
gtgatgttgc
atgatgctct
gtcagtcagt
tggcagacac
aacaactgga
gatgggcaac
gcctaaagta
aacattttct
agaaacgaag
aacagtaaac
catttttttc
aaattaaaca
tgtcaggtgc
ggaacttctc
tacacgtatt
tttagcgtgg
caaaggtata
ctttagcaag
aggacgtttc
cttttattaa
agtaaatata
aatcttttcg
ccttccatgt
ctgacctcct
agtattggtt
agatgtatgt
aaccgctaga
tgatccctcc
ttaccctgat
aattgaggca
ctgatcacca
tctgtcactg
aagtgaattt
acttttaatt
gacccaggct
ctgcaagtgc
gcaagatgca
tgggggagag
ccagcaagcg
tggtcacgca
tcaatcgcag
caacatatta
atgggttaat
cgcctagttg
tgcaaaatgc
ggatttttta
gagcctggtt
ccggatcacc
gccgtagcca
tgaccttcgt
tgcatatcgg
gtaatcgacc
gatgccagaa
ggcaatcctt
aaaaacagta
cgacttcgcc
cggtactgac
agatggtaga
gaactgataa
agctatttac
aatcaacagg
agcttggcct
aggagcgtgg
gcagcaatat
acagcctgat
cagagtcacc
tgttaatcat
agaactgaag
tgcgctcgat
gcgtgatgat
gcgtgaagcc
cgctgaacgg
aggaacccag
tcatgcaatt
ataaaaccga
gcgccgccac
aaatgatggg
gtctgttgag
atggtgttat
aaccctaaac
gatgaatcgt
tgcgggagtg
gcattatgcc
aaagatttgt
gtaatatctt
attttccctg
tataagatgc
cacggtgtta
atgtgagacg
cacttgatcg
gatacgaggg
tgtgttttgt
gcgtaacaaa
aaggccaacg
tgaagagcaa
gtggatctga
gttgtaattg
gcgttggtga
taactgctaa
tcaggaaagt
acaatatcgt
gatgtatatg
gagaaattcc
tcgcaacatt
gagattgcca
ttgtcgagaa
cagcatatcg
ctgttaagcc
acaacatttt
acggcatgat
tcgctcgttg
gtcacttcga
aatcccgaaa
tatctgcaca
agccagtgct
aaatgcgtac
ctgtctgtcc
gaaagcgggt
tcacgaacaa
ttattcctaa
aaacatgacc
gcgtttgcaa
atcgacgcaa
ggactaagta
tcgattggtt
aatcaataat
cggacgtcag
tgattactcc
cgccggacgc
gaaagacttc
cgctttacct
ctgggcttca
tgcaaaattc
gcgattatct
taccgtgata
ctggcgaacg
gcaaaataca
gttgccgctg
accaccgcct
gattatttca
aagtatatta
attgtgagca
gcaatccatt
aaattttggc
tgatggtttc
cacatcctgt
tcccgatgct
aatgagttga
cattgtattc
tccgggaata
aacgccccgg
gtagtgttct
ttatgttcat
tattgctgaa
gtgtttcttg
tcgttttcta
ttgtgacgtt
aatatttctt
cgcgtagttt
tgatgattta
gtgcggtcct
tgctcaaatc
gcgcatggag
ttcgtgtaaa
catgtataga
agcacgataa
tcattcaaac
ggtaaaactg
cctgttcgga
ctctcttttc
cggacccttt
cgcttatgcg
tggtacaggc
agagtgcgga
cgctgtgacg
gctgtatgac
gaatgcggtc
attgacttat
tggtagtgag
cgtatcgtct
cagttcgcag
acaggtaaga
ctttccgttg
aggcgtcatc
tgaattcatt
ggcaggaggt
atctgattac
ttaaatagag
tgttggccgc
tggcgtacct
cgatgtgcgc
gcaatctcgc
cgcttatcaa
caacgtaagg
aaaaccagaa
gatcaccctc
taccagcttc
tctccgaaaa
atgattgatc
ctgccgggcg
aaagaagcgg
ccgctctggt
acgccattac
cggcaattac
cgaaggagtt
gtcgtcgtcg
ccggcgtgga
ccctcagaga
atgagcagtg
atacacacgc
tacgaatgtt
tgcatcgaca
ctttggtgct
aataagcagg
ttttgaagtt
aatttcatat
ccggattaac
attaaaacga
tgctgacacg
gaatgctctc
ggatatttgt
atgtgatttc
agaatttaac
acacgatgtg
ttagttcaga
taaaaatggc
gcattatcgt
tgtcaaatat
gctggcattc
ttcatacaga
cgacaaaatg
aaatatgctt
acataaggtg
taatatgaag
tatttagtct
caactcaatt
gggaagaacg
tgacgttagt
ttgctcaaga
gattattgcc
cgtgcggttg
agatgcaaag
atgctaatcc
gctctggtgg
acacgttagc
tgaataaaat
atgaaaagag
ggaactccaa
gtaatagtta
gcattgagtc
tgctgaatta
gccgcccagc
agtaatagtt
cgcgctaaca
taaacacagt
caaatcccct
cattctcgcg
tcgcggcaga
cattatcgcc
ttatataacg
acgcttcgct
cgttcctcga
atcatggtta
gcaaacttgt
tttcccgttg
gtcaggacgc
gtggtgatat
ctggttatgg
gcggaacggt
tatctgcatc
ctacaaagcc
tgacatgcag
agctgatgct
gttgcacatc
taatgcagcc
gaggctgatc
cagatagagt
gcttccagcg
tgctgggttt
gttttcttct
actgctgccg
gccagcgcag
cgcagaatcg
tgttaatatt
tatgtccaca
tgcacacagg
gaagaaaccg
agtaaatagt
aacccatcgg
tcttgatttc
atttacaacc
aatattatct
ataaaacaat
aacctgagcc
ttttatcgtt
taggaatgtt
tggagggaaa
aagatttgaa
aataaagaac
aatagcacca
tctctggaag
gattattccc
gtgacagagc
actgcaatgc
cgggatgttc
ctccgacggc
gcgatgttaa
gtagtgccgc
atattgccaa
gcgtcggcta
caaaccttac
tgcaatgcca
agcatgattg
tgggtaaatt
gcggcgctta
ccatcgcagg
gagcctgcat
gataatcgtg
agcgaatacc
aacagcacaa
acgctgcggc
acctcctgcc
agcctggatt
tattgggggt
gcaaaggaac
tataatggcg
tggttcattc
agcgtgttta
gctaaaaaag
tatgctggcg
tgacgtcatt
cacgctaaac
gtgggatgcc
tgtggcattg
ccgtcaggca
tcagttcgag
cagagagatt
atcgtctgcc
cagcgcgaca
atgcgtcagc
aaagctgaaa
aaagcagtct
tccccccgac
actatgcaaa
tgcccatatc
gagtataaat
ctgttttaac
gcccaattcc
gtttgttttg
tagcgagtag
tatgtgtaga
tattaatgta
gccctgacgg
gtttagcgcg
gacgttatga
aatgaattat
aaaactcctg
aacctatcat
tttttaagtc
gtggctagat
tcacagtcta
attggtaaaa
tcaatctggt
ttcacttaat
tacaaccgac
gtaatatttt
aatctgctga
tttctatgag
cattcagagc
tggtggttga
caacacgcag
cctcgtaatt
attcttcatc
aggcttcaat
tttgttcaat
48
48241
48301
48361
48421
48481
catttggtta
catgaggttg
aagttgatgc
gatggcctcc
tccggtgatc
ggaaagcgga
ccccgtattc
agatcaatta
acgcacgttg
cgacaggtta
tgttgcgggt
agtgtcgctg
atacgatacc
tgatatgtag
cg
tgttgttctg
atttgtattg
tgcgtcataa
atgataatca
cgggttctgt
tctgaagttg
ttgattattt
ttatcacttt
tcttcgttga
tttttacgtt
gacgtggttt
acgggtcctt
49
Appendix II
50
The MANCHESTER METROPOLITAN UNIVERSITY
COSHH RISK ASSESSMENT
FACULTY
SCHOOL OF BIOLOGY, CHEMISTRY
and HEALTH SCIENCE
Science and Engineering
DIVISION:
ACTIVITY
Cloning of lambda phage DNA in E.coli and the frequencies of the transferred genes
REASONS FOR ACTIVITY
Final year project
STATUS OF PERSONS UNDERTAKING ACTIVITY
Student Name (if applicable): Bozena Palinkasova
Student Course and Level (if applicable): BSc (Hons) Biology, stage 3
Supervisor/Staff Name and Status: Mr Howard Hughes
Specific location (Room Number) where work is to be carried out: T4.01
HAZARDOUS SUBSTANCES AND MATERIALS USED AND HAZARD
CLASSIFICATION (Continue on a separate sheet if necessary)
HAZARD NAME
HAZARD
R/S No.
RISK/SAFETY/HAZARD
LABEL
if available
PHRASES
Escherichia coli
Low risk infection
NM522, JM 101 Class 2 bacteria
(antibiotic attenuated).
Nutrient Broth
Nutrient Agar
plates Containing 50μg/ml, 80 μg/ml
methicillin X-gal & IPTG
(Qiagen Kit Extraction
hazard.
Risk & Safety
Phrases-NONE
LISTED
Risk & Safety
Phrases-NONE
LISTED
R22-36/38
May cause skin irritation.May be harmful if
absorbed through the skin. May cause eye
irritation.Material may be irritating to mucous
membranes and upper respiratory tract. May be
harmful if inhaled. May be harmful if
swallowed.(Sigma)
Ampicillin, Methicillin)-Irritating to eyes,
respiratory system and skin. May causesensitization
by inhalation and skin contact May cause allergic
reaction.(Sigma)
X-gal-( 5-Bromo-4-chloro-3-indolyl β-DgalactopyranosideMay cause skin irritation. May be
harmful if absorbed through the skin. May cause
eye irritation Material may be irritating to mucous
membranes and upper respiratory tract. May be
harmful if inhaled. May be harmful if
swallowed.(Sigma)
IPTG (iso propyl β-d-thiogalactopyranoside)May cause skin irritation. May be harmful if
absorbed through the skin. May cause eye
irritation. Material may be irritating to mucous
membranes and upper respiratory tract. May be
harmful if inhaled. May be harmful if swallowed.
(Sigma)
Contains guanidine hydrochloride, acetic acid:
Harmful if swallowed, irritating to eyes & skin.
51
Method)
Buffer N3
Buffer P2
Buffer PB
RNase A
Contains ribonuclease: sensitizer. .The
RNase solution is added to the P1
resuspension Buffer
Ligation Mix
(Qiagen)
S13-23-2636/37/39-46
Contains sodium
hydroxide: irritant.
Hazard Class;
Flammable
Contains guanidine
hydrochloride,
isopropanol: harmful,
flammable, irritant.
Irritating to eyes & skin.
R36/38
S13-23-26-36-46
(Risk Phrases
R10-22-36/38 &
Safety PhrasesS13-23-2636/37/39-46).
(RiskPhrases
R42/43 & Safety
Phrases- S23-2426-36/37).
– pUC 18 (approx 0.5 μg) + 1μg λ DNA
+ Ligation Buffer + 1μg T4 ligase
T4 Ligase, λ DNA
Risk & Safety
Phrases-NONE
LISTED
pMAL – c2X
Risk & Safety
Phrases-NONE
LISTED
Risk & Safety
Phrases-NONE
LISTED
T4 DNA ligase
Alkaline phosphatase,
Calf Intestinal
Risk & Safety
Phrases-NONE
LISTED
Lambda DNA
Risk & Safety
Phrases-NONE
LISTED
Hind III
Risk & Safety
Phrases-NON
LISTED
Ethidium Bromide
R22-26-36/37/3868,
S26-28-36/37-45
Loading buffer :
Bromophenol Blue(0.25% in 100ml)
Orange G
Xylene CyanolFF
Risk & Safety
Phrases-NONE
R – NONE
S24/25
1 kb plus DNA Ladder
Electrophoresis running
buffer
Conc X10 (diluted 1 in 10 for class use).
To the buffer at normal strength 0.5μg/ml
ethidium bromide may be added to help
May cause sensitization by inhalation and skin
contact
Prolonged or repeated exposure may cause allergic
reactions in certain sensitive individuals. May
cause skin irritation. May be harmful if absorbed
through the skin. May cause eye irritation. Material
may be irritating to mucous membranes and upper
respiratory tract. May be harmful if inhaled. May
be harmful if swallowed.(Sigma)
May cause skin irritation. May be harmful by
inhalation, ingestion, or skin absorption. May cause
eye irritation.(New England BioLabs)
May be harmful if absorbed through the skin. May
cause eye irritation. Material may be irritating to
mucous membranes and upper respiratory tract.
May be harmful if inhaled. May be harmful if
swallowed.(New England BioLabs)
May be harmful if absorbed through the skin. May
cause eye irritation. Material may be irritating to
mucous membranes and upper respiratory tract.
May be harmful if inhaled. May be harmful if
swallowed. ( New England BioLabs)
May cause skin irritation. May be harmful by
inhalation, ingestion, or skin absorption. May cause
eye irritation.(Invitogen)
May cause skin irritation. May be harmful by
inhalation, ingestion, or skin absorption. May cause
eye irritation.(Fermentas)
Harmful by ingestion. Irritating to skin, eyes and
respiratory system. Very toxic if inhaled. Possible
risk of irreversible effects. Evidence of mutagenic
effects. (Sigma)
May cause skin irritation, may cause eye irritation.
May be harmful by inhalation, ingestion or skin
absorption, possible allergen.(Promega)
+TBE buffer X 10
LISTED
Agarose :
0.8% gel made up in single strength
electrophoresis buffer to which is added
for use 0.5μg per ml ethidium bromide.
Harmful if swallowed, irritating to eyes & skin.
(Qiagen)
R36/37/38
S26, S37/39
May cause skin irritation. May be harmful if
absorbed through the skin. May cause eye irritation.
Material may be irritating to mucous membranes &
upper respiratory tract. May be harmful if inhaled.
May be harmful if swallowed. (Sigma)
May cause skin irritation, may cause eye irritation.
May be harmful by inhalation, ingestion or skin
absorption. (Invitrogen)
Tris Base 54g per litre made up
May cause skin irritation, may cause eye irritation.
May be harmful by inhalation, may cause severe
gastrointestinal irritation with nausea & vomiting.
May cause liver & kidney damage. May cause
metabolic changes including hypoglycaemia &
52
increase the intensity of the DNA bands
hyperkalemia. May depress the respiratory centre
skin absorption. Prolonged or repeated exposure
may cause allergic reactions in certain sensitive
individuals. (Fisher)
R61, R60
S53,S45
R36/37/38
S26 S37/39
Orthoboric acid (Boric acid)- Hazard Class –
Harmful. 27.5g per litre used
Irritating to eyes. Harmful by ingestion., skin
contact , & if inhaled as a dust. Can be absorbed by
the skin if the skin is cut or abrased. Causes
gastrointestinal irritation with nausea & vomiting &
diarrhea. Causes kidney damage, with red skin rash
followed by extensive exfoliation not only in areas
of rash but also of mucous membranes. Other
symptoms may include weakness, headache &
restlessness. Prolonged or repeated exposure may
cause dermatitis. Evidence of reproductive
effects.(Fisher)
Disodium ethylenediaminetetra acetic acid –
(EDTA) 20ml of stock 0.5mol dm—3 added per
litre
Causes eye & skin irritation. Cause irritation of the
mucous membranes & upper respiratory tract. May
cause gastrointestinal irritation with nausea,
vomiting & diarrhoea. Exposure may cause kidney
injury, muscle cramps, bone marrow depression,
and a generalized allergic reaction. May cause
reproductive & foetal effects. Possible mutagen.
Chronic exposure may cause kidney damage.
(Fisher)
ALL CONTAINERS OF HAZARDOUS SUBSTANCES SHOULD BEAR THE CORRECT HAZARD
WARNING LABELS
53
RISK ASSESSMENT
EXPOSURE CONTROLS / PERSONAL PROTECTIVE EQUIPMENT
SAFETY SPECTACLES (conforming to BS EN166) AND LABORATORY COAT TO BE WORN AT ALL TIMES
Wash your hands thoroughly before leaving the laboratory
FIRE
IF A FIRE BREAKS OUT YOU SHOULD INFORM A MEMBER OF ACADEMIC STAFF OR
TECHNICIAN IMMEDIATELY
FIRST AID
ALL ACCIDENTS, OR INCIDENCES OF FEELING UNWELL, MUST BE REPORTED TO A
MEMBER OF ACADEMIC STAFF OR TECHNICIAN IMMEDIATELY. IF ADVERSE EFFECTS ARE
NOTED AFTER THE LABORATORY SESSION OBTAIN MEDICAL ATTENTION IMMEDIATELY
Eye Contact
IN ALL INSTANCES EXCEPT MINOR SKIN CONTAMINATION SEEK MEDICAL ATTENTION
Irrigate thoroughly with water for 10 minutes. (Irrigation stations are available at every sink)
Inhalation
Remove from exposure rest & keep warm.
Skin Contact
Drench skin with water & remove contaminated clothing.
Ingestion
Wash out & give plenty of water to drink.
SPILLAGE
IN CASE OF LARGE SPILLAGE OF CHEMICALS CONSULT A MEMBER OFACADEMIC STAFF
OR TECHNICIAN. SMALL SPILLAGE MAY BE TREATED ACCORDING TO THE DIRECTIONS
GIVEN BELOW:
(there is a spill kit available in room 4.01B + Algosol Spill absorbent )
Spillage of all reagents should be dealt with by washing area with copious amounts of water & drying with
paper towels. Spillages of any solid material should be scooped up & dissolved in water, & the solution poured
down the drain with plenty of water.
Spillages of the microbial cultures- the area should be swabbed down with suitable decontamination fluid. (E.g.
70% Propan-2-ol or 2.5% sodium hypochlorite.
DISPOSAL
Ethidium bromide residues can be neutralised by mixing with bleach to a non toxic non mutagenic compound
that can be poured down the sink with lots of water- 34mg ethidium bromide to 100ml bleach stirred 4 hours
before disposal ( TBE- buffer can be remixed & used a number of times before disposal )
All other solutions can be disposed of down the drain with plenty of water. All waste agarose solution should
be boiled up & diluted before disposal to the sink with a large quantity of water to avoid blockage of the sink
drains.
The microbiological cultures should be collected up for autoclaving before disposal. All bacterially
contaminated glassware e.g. pipettes, should be placed in suitable receptacle with disinfectant. All disposable
plastic ware & paper e.g. tissues, should be placed in an autoclave bag & autoclaved.
HAZARDOUS MATERIALS MUST NOT BE REMOVED FROM THE LABORATORIES
DATE OF ASSESSMENT
SIGNATURES OF ASSESSORS
Student:
Supervisor/Staff:
DATE TO BE REVIEWED BY:
SIGNATURE OF HEAD OF DIVISION
SIGNATURE OF HEAD OF SCHOOL
Division Health & Safety Coordinator:
BCHS SCHOOL REFERENCE NUMBER:
54
Declaration:
With the exception of any statements to the contrary, all the data presented in this report
are the result of my own efforts. In addition, no parts of this report have been copied
from other sources. I understand that any evidence of plagiarism and/or the use of
unacknowledged third party data will be dealt with as a very serious matter.
Signed .................................................................
Date ........................
55
ASSESSMENT PRO-FORMA
A – Log Book
Mark to be assigned by the project supervisor
Mark awarded (out of 100)
B – Effort, application, innovation & skills
Mark to be assigned by the project supervisor only
Mark awarded (out of 100)
C - Project Report
Mark to be agreed between the project supervisor and second marker
Mark awarded by supervisor
(out of 100)
Mark awarded by second marker
(out of 100)
Agreed mark
(out of 100)
Overall project mark
(20% A + 20% B +60%C )
88%
56