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A Plasmid Cleaved by Restriction
Enzymes
Námsbraut: Raunvísindadeild; Líffræðiskor
Námskeið: 09.51.35 Erfðafræði
Nafn kennara: Ólafur S. Andrésson, Zophonías O. Jónsson, Sigríður H. Þorbjarnardóttir og
Bryndís K. Gísladóttir.
Vikudagur, hópur: Fimmtudagur, síðari kennslustund, Hópur 2 og 4.
Tilraun framkvæmd: 09., 16. október, 2003
Skýrsluskil til kennara: 13. nóvember, 2003
Skýrsluskil til stúdenta:
Einkunn:
Nöfn stúdenta: Bjarki Steinn Traustason, Egill Guðmundsson, J. Gabriel-Rios Kristjánsson,
Marcella Manerba og Nicoletta Palmegiani.
________________________________
Bjarki Steinn Traustason
________________________________
J. Gabriel-Rios Kristjánsson
________________________________
Egill Guðmundsson
________________________________
Marcella Manerba
________________________________
Nicoletta Palmegiani
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9
Erfðafræði
A Plasmid Cleaved by Restriction Enzymes
Introduction:
When one wants to genetically modify an organism, a so-called vector is usually used
for the process. The usage of plasmids, a type of vector, is most frequent, which are
small double stranded DNA molecules. The transfer operation of these plasmids into a
bacterium’s chromosome, is fairly easily done. Plasmids account for about 5% of the
bacterial genome, and they are able to replicate outside the bacterium’s chromosome.
The chromosome features, amongst other things, the genes, that make it resistant to a
number of chemicals.
In eucaryote cells, plasmids come to pass very seldom. So-called restriction
enzymes, make the insertion, of a desired gene in to a plasmid, possible. They
identify specific nucleotide DNA molecules, usually of 4 to 6 bp segment and cleave
the strand within those sequences. The same restriction enzyme specifically applies to
the plasmid, as to the, becoming, inserted DNA strand. The both ends of open circular
plasmid are complimentary to both ends of the DNA segment, respectively. The
segment is inserted into the circle with the help of intergrating enzymes.
In this practical experiment, R21-segment was integrated, via HindIIIrestriction site, into the pUC18 plasmid (2700 bp), subsequently called pUC18-R21.
Then, pUC18-R21, was cleaved, separately, by four restriction enzymes, and the
segments formed, were electrophoresised in agarose gel, and finally the results
photographed (FIGURE 02 ■). The enzymes used, cleave the pUC18 plasmid, just once
or never (XhoI).
Aims/Hypothesis:
The aim of this exercise, is to evaluate the size of the integrated DNA segment, and to
delinate schematic drawing of the pUC18-R21, and designate, where the restriction
enzymes cleave it.
Design and Methods:
Reference to work sheets in manual booklet, for present exercise (p.39-42).
Exception in the procedure part, p.39, where intermixture for each group (H1H6) was the following (TABLE 01 ■):
TABLE 01, SHEME FOR INTERMIXTURES:
H1
H2
H3
H4
H5
H6
pUC18-R21
[µL]
Buffer
[µL]
Restriction enzymes
7 µL of ..
Well no.
2.0
2.0
2.0
2.0
2.0
2.0
7.5
7.5
7.5
7.0
7.0
6.5
HindIII
PstI
SacI
SacI and HindIII
XhoI
XhoI and PstI
4
5
6
7
8
9
Results:
The information about the size of pUC18 plasmid was atteind from www.neb.com, ie,
2.7 kb. Also, knowledge gained from the internet, which of our restriction enzymes
cleave only the pUC18 plasmid, once, but never R21. → They are; HindIII at 399th
bp; PstI at 415th bp; and SacI at 448th bp. (FIGURE 01 ■)
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Based on this information, one can assume that, if segment formed, are more
then one, using each of the enzymes, they also cleave R21-insertion. Also, XhoI, does
not cleave pUC18 palsmid, then one can assume, XhoI only lceaves R21, if the result
of electrophoresis gives linear segment the same size as linear pUC18-R21.
FIGURE 01, pUC18 WITH LOCATION OF SOME RESTRICTION SITES:
2.7 // 0
0.399, HindIII
0.415, PstI
0.448, SacI
The numbers of lengt, given in the figure, are in kb.
□
FIGURE 02
■, is a photo of the results form electrophoresis. Into wells no. 01 an 10, the
markers: lambda HindIII and pATX Hinf; and 100 kb DNA ladder; respectively, were
placed. Everything about these markers, is known, making them useful to mark a
scalar into the photo.
FIGURE 02, ELECTROPHORESIS:
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10
More infromation is found in TABLE 02■.
□
TABLE 02, INFORMATION FOR ELECTROPHORESIS:
Column:
Plasmid:
Restriction E.:
Size of segment:
Size of segment:
Size of segment:
2
3*
4
5
6
7**
8
9
pUC18 pUC18-R21 pUC18-R21 pUC18-R21 pUC18-R21 pUC18-R21 pUC18-R21 pUC18-R21
HindIII none
HindIII PstI
SacI
SacI+HindIII XhoI
XhoI+PstI
~ 2.7
~ 3.2
~ 2.9
~ 5.6
~ 3.4
~ 2.7
~ 5.6
~ 5.1
~ 2.7
~ 2.2
~ 2.1
~ 0.55
~ 0.74
*This is circular particle. It´s size (3.2 kb) is irrelevant, since circular particles travel for longer distance in electrophoresis,
compared to linear praticles.
**Based on subsequent results, a very small segment of ~ 48 bp in length, is also formed. But hue to it’s relatively small size, it
does not come to pass in this electrophoresis.
FIGURE 03■,
Illustrates given information for pUC18-R21, summarised.
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FIGURE 03, SUMMARISED; SEGMENTS FORMING pUC18-R21:
The most central ring represents as pUC18-R21,
where the yellow band illustrates R21-insertion. The
other six rings, around this central one, signify the
different lengths of segments formed from pUC18R21. Also, these six rings are marked from C4 – C9,
representing columns 4 – 9 in electrophoresis. The
numbers of length, given in the figure, are in kb.
SacI
~5.1
0.74
5.6
~2.7
3.4
5.6
2.7
2. 7
□
0.399
// 0 HindIII
C4 C5 C6 C7 C8 C9
0.44
8
Sa c I
0.415
PstI
0
0.40 III
Hind
Conclusion/Discussion:
The length of the insertion (R21) is
2.2
worked out according to compari2.1
son of known lengts of segments.
Those numbers of lengths are
0.05
obtained from the data in FIGURE 03
0.55
■, but naturally there is a signiXhoI
ficant error in them. Summarising
those information, in addition to information attained from www.neb.com and from
manual booklet, one can estimate the order of restriction sites in pUC18-R21, in
relatively accurate manner.
These methods, used in this project, are both popular and prespicuous,
exrecised in many fields of molecular biology.
2.9
Answers to Questions:
1.
The pUC18-R21 has the total lengt of ~ 5.6 kb. The pUC18 plasmid is 2686
bp long (~ 2.7 kb), resulting in the size of the insertion, R21, to be ~ 2.9 kb.
(FIGURE 03 ■).
2.
SacI and XhoI are the restriction enzymes that cleave the R21, each cleaving it
once. (FIGURE 03 ■).
3.
FIGURE 04
■.
FIGURE 04, THE MAP OF pUC18-R21:
HindIII, 0.399, 0.0
0.0
SacI, 0.74
2.7
The ring represents as the pUC18-R21, the yellow band
illustrates the R21-insertion, and the rest, pUC18. The numbers
of lenght are in kb. Numbers coloured red, signify kb for R21.
Numbers coloured black, signify kb for pUC18. Data was
obtained from FIGURE 02 ■.
□
PstI, 0.415
SacI, 0.442
XhoI, 2.4
HindIII, 0.400, 2.9
■
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