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GeneJET™ PCR Cloning Kit
COMPONENTS OF THE KIT
#K1221 for 20 reactions
#K1222 for 40 reactions
Component
For 20 reactions
(#K1221)
For 40 reactions
(#K1222)
TABLE OF CONTENTS
pJET1/blunt Cloning Vector (50ng/µl)
24µl
46µl
COMPONENTS OF THE KIT ..................................................... 2
2X Reaction Buffer
240µl
460µl
T4 DNA Ligase (5u/µl)
24µl
46µl
DNA Blunting Enzyme
24µl
46µl
pJET1 Forward Sequencing Primer,
10µM aqueous solution
5'-gcctgaacaccatatccatcc-3',
50µl
100µl
pJET1 Reverse Sequencing Primer,
10µM aqueous solution
5'-gcagctgagaatattgtaggagatc-3
50µl
100µl
Control PCR Product (24ng/µl)
976bp, with 3’-dA overhangs
8µl
12µl
Water, nuclease free
1.5ml
1.5ml
STORAGE ............................................................................. 2
INTRODUCTION......................................................................2
GeneJET™ CLONING PRINCIPLE ..............................................3
PCR PRODUCT CLONING WORKFLOW .....................................3
CLONING PROTOCOL .............................................................3
Ligation with pJET1/blunt cloning vector ............................ 4
Transformation .................................................................6
Analysis of recombinant clones .........................................6
CONTROL EXPERIMENT..........................................................7
QUALITY CONTROL ................................................................ 8
STORAGE
MAP AND FEATURES OF pJET1/blunt CLONING VECTOR ..........8
Genetic elements of the pJET1/blunt Cloning Vector ...........9
All components of GeneJET™ PCR Cloning Kit should be stored at -20°C.
DNA Sequence of MCS region........................................... 9
INTRODUCTION
Restriction enzymes that do not cut pJET1/blunt ............. 10
Restriction enzymes that cut pJET1/blunt once ................ 10
Primer sequences ..........................................................10
PREPARATION OF LB-AMPICILLIN PLATES .............................10
TROUBLESHOOTING.............................................................11
RELATED PRODUCTS ...........................................................13
References ..........................................................................14
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™
The GeneJET PCR Cloning Kit is an advanced positive selection system for the highest
efficiency cloning of PCR products generated with Pfu DNA Polymerase, Taq DNA polymerase
or any other thermostable DNA polymerase. Additionally, any other DNA fragments, either
blunt or sticky-end, can be cloned using this kit. Purification of PCR products prior to cloning
is not required. The cloning efficiencies are up to 100% , thus eliminating the need for tedious
colony screening.
A novel, high-copy, ready-to-use positive selection cloning vector, pJET1/blunt, is included in
the kit. The vector contains the gene for a restriction endonuclease which is lethal for all E.coli
strains commonly used for cloning. Ligation of a DNA fragment into the cloning site disrupts
this lethal gene. As a result, only cells with recombinant plasmids are able to propagate.
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GeneJET™ CLONING PRINCIPLE
The pJET1/blunt is a linearized blunt-end cloning vector, ready for ligation with blunt or blunted
PCR products or other blunted DNA fragments. The 5'-ends of DNA at the vector cloning
site contain phosphoryl groups. Therefore, ligation of a blunt PCR product does not require
additional phosphorylation of the PCR product or the use of phosphorylated primers during the
PCR reaction. Blunt-end PCR products generated by proofreading DNA polymerases can be
directly ligated in just 5min with the pJET1/blunt cloning vector.
PCR products with 3'-dA overhangs generated using Taq DNA polymerase or other nonproofreading thermostable DNA polymerases are blunted for 5min with a highly efficient
thermostable DNA Blunting Enzyme (included in the kit) prior to ligation. The Reaction Buffer is
optimized both for blunting and for ligation reactions.
The pJET1/blunt is a positive selection vector. Therefore, only recombinant plasmids containing
the insert DNA are propagated in the E.coli cells. Recircularized pJET1/blunt vector molecules
lacking the insert express a lethal restriction endonuclease after transformation and are not
propagated. This feature drastically accelerates the process of colony screening. Since up
to 100% of colonies contain the recombinant plasmids, the need for blue/white screening is
eliminated resulting in saving of expensive reagents, such as IPTG and X-Gal.
PCR PRODUCT CLONING WORKFLOW
1.
2.
3.
4.
Generation of a blunt-end PCR product.
Ligation of the PCR product into the cloning site of pJET1/blunt.
Transformation of competent E.coli cells with the ligation reaction mixture.
Analysis of recombinant clones for presence and orientation of the insert.
CLONING PROTOCOL
Important Notes
• Thoroughly mix every vial before use.
• Gel-analyze the PCR product for size and yield before cloning.
• Purification of the PCR product is not required if a specific product has been generated
during PCR.
• Cloning of a PCR product which is contaminated with primer-dimers and/or with nonspecific PCR products may result in a lower number of target clones. In these cases,
gel purification of the desired amplicon is recommended (e.g. using DNA Extraction Kit
#K0513 or Agarase #EO0461).
• If the PCR template encodes ß-lactamase (e. g., ampicillin resistance gene), background
colonies on LB-ampicillin agar plates may appear. Gel purification of the PCR product is
highly recommended when using such templates.
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• For efficient cloning of gel-purified DNA fragments, care should be taken to avoid DNA
damage with UV light. Always use a long wavelength UV (360nm) light-box during excision
of the agarose gel slice. If only a short-wavelength (254-312nm) light-box is available,
minimize the UV exposure to several seconds. To avoid DNA exposure to UV altogether,
visible dyes can be included in standard agarose gels to visualize DNA bands in ambient
light (1, 2).
• The GeneJET™ PCR Cloning Kit is compatible with all PCR buffers supplied by Fermentas.
• The kit performs well over a wide range of insert/vector molar ratios (0.5:1 to 15:1). The
optimal insert/vector ratio is 3:1.
Ligation with pJET1/blunt cloning vector
• Follow the Blunt-End Protocol for cloning blunt-end PCR products generated with Pfu
DNA polymerase, or with other proofreading DNA polymerases.
• Follow the Sticky-End Protocol for cloning PCR products with 3'-dA overhangs
generated with Taq DNA polymerase, or with enzyme mixes containing Taq DNA
Polymerase (e.g Long PCR Enzyme Mix or High Fidelity PCR Enzyme Mix from Fermentas).
• If the end structure of PCR products is not specified by the supplier of DNA polymerase, it
is recommended to follow the Sticky-End Protocol.
• Do not use more than 2.0 µl of the PCR mixture in the ligation reaction to avoid inhibition
of T4 DNA ligase by salts present in the PCR buffer.
Blunt-End Protocol
For cloning blunt-end PCR products generated by proofreading DNA polymerases.
1. Set up the ligation reaction:
Component
2X Reaction Buffer
PCR product (non-purified)
pJET1/blunt Cloning Vector (50ng/µl)
Water, nuclease-free
T4 DNA Ligase
Total volume:
Volume
10µl
1-2µl
1µl
up to 19µl
1µl
20µl
Vortex briefly and centrifuge for 3-5 sec.
2. Incubate the ligation mixture at room temperature (22°C) for 5min.
Note. Incubation time can be extended up to 30min if the maximal number of
transformants is required.
3. Use the ligation mixture directly for bacterial transformation.
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Sticky-End Protocol
For cloning PCR products with 3’-dA overhangs generated using
either Taq DNA polymerase, or enzyme mixes containing Taq DNA Polymerase.
1. Set up the blunting reaction:
Component
2X Reaction Buffer
PCR product (non-purified)
Water, nuclease-free
DNA Blunting Enzyme
Volume
10µl
1-2µl
up to 17µl
1µl
Total volume: 18µl
Vortex briefly and centrifuge for 3-5s.
2. Incubate the mixture at 70°C for 5min. Chill on ice for several seconds.
3. Set up the ligation reaction. Add the following to the blunting reaction mixture:
Component
Volume
pJET1/blunt Cloning Vector (50ng/µl)
1µl
T4 DNA Ligase (5u/µl)
1µl
Total volume: 20µl
Vortex briefly and centrifuge for 3-5s.
4. Incubate the ligation mixture at room temperature (22°C) for 5min.
Note. Incubation time can be extended to 30min to obtain the maximal number of
transformants.
5. Use the ligation mixture directly to transform competent E.coli cells.
Transformation
The GeneJET™ PCR Cloning Kit is compatible with the most E.coli laboratory strains.
Transformation of competent E.coli cells with the ligation mixture can be performed using a
number of different transformation methods. Use competent E.coli cells with a transformation
efficiency of at least 1x106 transformants per µg supercoiled plasmid DNA.
If using commercially-supplied competent cells, follow the recommendations from the supplier.
For fast cloning, we recommend using competent cells prepared with the TransformAid™
Bacterial Transformation Kit (#K2710).
Transformation tips:
• Use up to 2.5µl of the reaction mixture to transform 50µl of competent E. coli cells
prepared with the TransformAid™ Bacterial Transformation Kit (#K2710).
• To transform competent E. coli cells prepared by the calcium chloride method, use up to
5µl of the ligation mixture per 50µl of competent cells;
• For transformation by electroporation, extract the ligation reaction mixture with chloroform.
Then, use a 1µl aliquot of the extracted ligation mixture to transform 50µl of competent
cells.
Transformation of competent E.coli cells prepared with TransformAid™ Bacterial
Transformation Kit
1. Prepare LB-ampicillin agar plates (without IPTG and X-Gal, see p. 10 ). Pre-warm the
plates at 37°C for at least 20min.
2. Prepare competent E.coli cells as described in the protocol provided with the
TransformAid™ Bacterial Transformation Kit.
3. Transfer 2.5µl of the ligation mixture into a new microcentrifuge tube. Chill 2min on ice.
4. Add 50µl of the prepared competent E.coli cells. Incubate 5min on ice.
5. Plate immediately on pre-warmed LB-ampicillin agar plates. Incubate overnight at 37°C.
Analysis of recombinant clones
Analyse 4-6 colonies for the presence and orientation of the DNA insert using one of the
following methods:
1. Restriction analysis. Isolate plasmid DNA from an overnight bacterial culture using a
convenient plasmid miniprep method. To speed up the process and to assure the quality
of purified plasmid DNA, use the GeneJET™ Plasmid Miniprep Kit (#K0501). To digest DNA
from recombinant clones in just 5 minutes, use FastDigest™ restriction enzymes.
2. Colony PCR. Use the pJET1 Forward Sequencing Primer and the pJET1 Reverse
Sequencing primer supplied with the kit in the PCR.
3. Sequencing. Use the pJET1 Forward Sequencing Primer or pJET1 Reverse Sequencing
Primer supplied with the kit for sequencing of the cloned insert.
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CONTROL EXPERIMENT
To perform a control cloning experiment, use a 2µl (48ng) aliquot of the Control PCR product
provided with the Kit. The control PCR product has been generated with the Taq DNA
polymerase, which adds extra nucleotides to the 3’-end. Therefore, the Sticky-End Protocol
(PCR product blunting and ligation) is to be followed to control the efficiency of both blunting
and ligation steps.
1. Set up the blunting reaction:
Component
2X Reaction Buffer
Control PCR product (24ng/µl)
Water, nuclease-free
DNA Blunting Enzyme
of electrocompetent E.coli cells. Important: extract the ligation mixture with chloroform
prior to electroporation to remove ligase.
• Transformation efficiency of E.coli competent cells, prepared with the TransformAid™
Bacterial Transformation Kit (#K2710), generally exceeds 107cfu/µg when the cells are
transformed with the pUC19. Usually, such cells yield hundreds of transformants when
a 50µl aliquot of the competent cells is transformed with 2.5 µl of the ligation mixture.
QUALITY CONTROL
Volume
10µl
2µl
5µl
1µl
Total volume: 18µl
Each lot of the kit has been tested for efficient cloning of the 976 bp Control PCR Product.
A 2µl aliquot of the ligation mixture was used to transform 50µl of chemically competent
DH10B cells. Transformation efficiency of the cells with pUC19 was 0.5-1x107 cfu/µg. Cloning
efficiency of the Control PCR Product into the pJET1/blunt exceeded 1x105 cfu/µg . More than
96% of the recombinant pJET1/blunt plasmids contained the appropriate insert. Each lot of the
pJET1 Forward Sequencing Primer and pJET1 Reverse Sequencing Primer was functionally
tested in DNA sequencing and in colony PCR using the recombinant pJET1/blunt .
Vortex briefly and centrifuge for 3-5s.
2. Incubate the mixture at 70°C for 5min. Chill on ice for several seconds.
3. Follow with the ligation reaction. Add to the blunting reaction mixture:
Component
Volume
pJET1/blunt Cloning Vector (50ng/µl)
1µl
T4 DNA Ligase
1µl
Total volume: 20µl
MAP AND FEATURES OF pJET1/blunt CLONING VECTOR
The pJET1/blunt cloning vector is a pre-digested pJET1 vector (GenBank/EMBL Accession
number DQ317600). Blunt ends of the vector DNA containing 5'-phosphoryl groups were
generated using the Eco32I endonuclease.
Map of the pJET1/blunt Cloning Vector. Unique restriction sites within the multiple cloning site
are indicated on the Fig.1.
Vortex briefly and centrifuge for 3-5s.
4. Incubate the ligation mixture at room temperature (22°C) for 5min.
5. Transform competent E.coli cells with 1-2.5µl of the ligation mixture. Use a 2.5µl aliquot
to transform the chemically competent cells, or a 1µl aliquot of the chloroform extracted
ligation mixture to transform the electrocompetent cells.
6. Analyze 10 colonies for the presence of a 976 bp insert by the colony PCR with the pJET1
Forward Sequencing Primer and pJET1 Reverse Sequencing Primer. At least 9 of 10
colonies contain the plasmid with the 976 bp insert.
The number of transformants depends on transformation efficiency of E. coli cells (e. g., cfu/µg
DNA). Check the transformation efficiency of your competent cells by transforming 50µl of
cells with 0.1ng of a supercoiled circular plasmid, like pUC19. The transformation efficiency
should be at least 1x106 cfu/µg DNA.
• Electrocompetent cells generally provide the highest transformation efficiency
(>1x109cfu/µg DNA). Use a 1 µl aliquot of the ligation reaction mixture to transform 50µl
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Fig.1. pJET1/blunt vector map.
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Genetic elements of the pJET1/blunt Cloning Vector
Restriction enzymes that do not cut pJET1/blunt
Element
Function
Position (bp)
rep (pMB1)
A replicon (rep) from the pMBI plasmid is
responsible for the replication of pJET1
1294-1908
Replication start
Initiation of the replication
1308 +1
bla (ApR)
ß-lactamase gene conferring resistance to
ampicillin. Used for selection and maintenance of
recombinant E.coli cells
2068-2928
eco47IR
Lethal gene eco47IR enables positive selection of
the recombinants
187-879
PlacUV5
Modified Plac promoter for expression of the
eco47IR gene at a level sufficient to provide
positive selection without IPTG induction
895-1018
Multiple cloning site (MCS)
Mapping, screening and excision of the cloned
insert
545-470
pJET1 Forward Sequencing
Primer
Sequencing of insert, colony PCR
451-471
pJET1 Reverse Sequencing
Primer
Sequencing of insert, colony PCR
534-510
There are no restriction sites in pJET1 DNA for the following enzymes:
AarI, Acc65I, AjiI, AjuI, AlfI, BaeI, BclI, BcuI, BoxI, BplI, Bpu1102I, BseJI, BseRI, BsgI, BshTI,
Bsp68I, Bsp1407I, Bst1107I, BstAPI, BtgZI, Cfr42I, CpoI, CspCI, Eco81I, Eco91I, Eco105I,
Eco147I, EheI, FalI, FseI, FspAI, KpnI, KspAI, MlsI, MluI, Mph1103I, NdeI, OliI, PacI, PasI,
PauI, PdiI, Pfl23II, PsiI, Psp5II, PsrI, PsyI, SanDI, SdaI, SexAI, SfiI, SgfI, SgrAI, SgsI, SrfI, TstI,
Van91I, XagI, XcmI, XmaJI.
Restriction enzymes that cut pJET1/blunt once
AdeI
AloI*
ApaI
BamHI
BbvCI
BceAI
BcgI
BfiI
BglI
BglII*
BpiI
Bpu10I
BsaXI
BseYI
Bsp119I
Bsp120I
Primer binding sites:
DNA Sequence of MCS region
BspTI
BstXI*
Bsu15I*
BtgI*
BveI
CaiI
Cfr10I
Cfr9I
Csp6I
Eam1105I
Ecl136II
Eco130I*
Eco31I
Eco32I*
Eco47III
Eco52I
291
455
1030
1023
843
1734
2657
2186
2255
509
229
843
1101
1552
181
1030
141
466
543
534
390
1659
2221
1027
2620
2136
162
534
2208
495
1040
155
Eco72I
EcoRI
FaqI
GsuI
HincII
HindIII
Kpn2I*
LguI
MssI
MunI
Mva1269I
NcoI*
NheI
NotI
NsbI
PaeI
173
178
274
2226
168
750
469
1125
887
1018
848
534
146
154
2361
150
PdmI
PfoI
Ppu21I
PspXI*
PstI
PvuI
RsaI
SacI
SalI
ScaI
SmaI
TatI
XbaI*
XhoI*
XmiI
* – MCS
2736
46
173
477
1036
2508
2620
162
168
2619
1027
2619
503
478
168
Note. Enzymes in bold are produced by Fermentas.
Primer sequences
Primer
Sequence
pJET1 Forward Sequencing Primer, 21-mer
pJET1 Reverse Sequencing Primer, 25-mer
5’-GCCTGAACACCATATCCATCC-3’
5’-GCAGCTGAGAATATTGTAGGAGATC-3’
PREPARATION OF LB-AMPICILLIN PLATES
Note. Note that only fragments inserted into Kpn2I, XhoI, Eco32I, XbaI and BglII targets might be sequenced
with the indicated primers.
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Prepare Ampicillin stock solution (50mg/ml), store at -20°C.
Prepare LB-agar Medium (1 Liter): weigh out Bacto Tryptone® 10g, Bacto Yeast extract® 5g
and NaCl 5g. Dissolve solids in 800 ml of water, adjust pH to 7.0 with NaOH and adjust the
volume with water to 1000ml. Add 15g of Agar. Autoclave the medium.
Before pouring LB-ampicillin agar plates, allow the medium to cool to 55°C. Then, add 2ml of
ampicillin stock solution (50mg/ml) to a final concentration of 100µg/ml. Mix gently and pour
plates. Allow the LB-ampicillin agar medium to solidify.
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TROUBLESHOOTING
Problem
Solution
Few or no
transformants
Low transformation efficiency of competent E. coli cells.
Check transformation efficiency with 0.1ng of a supercoiled
vector DNA (e.g., pUC19). The competent cells should yield at
least 1x106 transformants per µg of supercoiled DNA.
Problem
Solution
Background
colonies that
contain plasmids
with incorrect
inserts
PCR products are contaminated with a template which
encodes ampicillin resistance.
Gel-purify the PCR product if the PCR template encodes a
ß-lactamase to avoid background colonies on LB-ampicillin agar.
Ligase was not removed prior to electroporation.
Make sure that the ligation reaction mixture was extracted with
chloroform prior to electroporation.
Incorrect protocol was used.
If Taq DNA polymerase, or any enzyme mix containing Taq DNA
polymerase, was used in PCR, always follow the Sticky-End
Protocol to blunt the PCR product prior to ligation.
T4 DNA Ligase was inhibited by salts present in the PCR buffer.
Do not use more than 2.0 µl of the PCR mixture in the ligation
reaction to avoid inhibition of T4 DNA ligase by salts.
Nuclease contamination.
Use only components provided with the kit. Nuclease
contamination (e.g., from low quality water) can impair the
integrity of the lethal gene, thus disabling positive selection with
the pJET1/blunt vector.
Increased number
of sequence errors
in the cloned insert
PCR product was damaged by UV light during excision
from the agarose gel.
Use a long wavelength UV (360nm) light-box when excising
DNA from the agarose gel. When using a short-wavelength
(254-312nm) light-box, limit DNA exposure to UV to several
seconds. Keep the gel on a glass plate or on a plastic plate
during illumination with UV. Alternatively, use dyes visible in
ambient light to visualize DNA in standard agarose gels (1, 2).
Background
colonies without
plasmid
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Non-specific PCR products, or primer dimers, were
cloned into the pJET1/blunt.
Gel-analyze the PCR product prior ligation with the pJET1/blunt.
If non-specific PCR products, or primer-dimers, were generated
during the PCR reaction, gel-purify the target PCR product.
Otherwise, optimize the PCR conditions to increase specificity.
Insufficient amount of antibiotic in agar medium.
Use 100µg/ml of ampicillin in LB-ampicillin agar plates. Allow
the LB medium to cool to 55°C before addition of the ampicillin
to it.
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PCR product was damaged by UV light during the
excision from agarose gel
Use a long wavelength UV (360nm) light-box when excising DNA
from the agarose gel. When a short-wavelength (254-312nm)
light-box is used, limit DNA exposure to UV to several seconds.
Keep the gel either on a glass or on plastic plate during UV
illumination. Alternatively, use dyes visible in ambient light to
visualize DNA in standard agarose gels (1, 2).
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RELATED PRODUCTS
Product
Pfu DNA Polymerase, native*
Pfu DNA Polymerase, recombinant*
Taq DNA Polymerase, recombinant, 5u/µl
2X PCR Master Mix
TrueStart™ Taq DNA Polymerase, 5u/µl
GeneJET™ Fast PCR Master Mix (2X)
High Fidelity PCR Enzyme Mix*
Long PCR Enzyme Mix*
dNTP Mix, 2mM each
dNTP Mix, 10mM each
dNTP Set, 100mM
TransformAid™ Bacterial Transformation Kit
GeneJET™ Plasmid Miniprep Kit
DNA Extraction Kit
*Not available in the USA.
Amount
100u
500u
100u
500u
100u
500u
2x1.25ml
100u
500u
2x1.25ml
100u
500u
100u
500u
1ml
5ml
0.2ml
1ml
5x1ml
4x0.25ml
4x1ml
4x5ml
20 reactions
40 reactions
25preps
50preps
250preps
100preps
References
Catalog #
EP0571
EP0572
EP0501
EP0502
EP0401
EP0402
K0171
EP0611
EP0612
K0211
K0191
K0192
K0181
K0182
R0241
R0242
R0191
R0192
R0193
R0181
R0182
R0186
K2710
K2711
K0501
K0502
K0503
K0513
1. Rand, K.N., Crystal Violet can be used to Visualize DNA Bands during Gel Electrophoresis
and to Improve Cloning Efficiency, Elsevier Trends Journals Technical Tips, Online, T40022,
1996.
2. Adkins, S., Burmeister, M., Visualization of DNA in agarose gels and educational
demonstrations, Anal Biochem., 240 (1), 17-23, 1996.
Trademarks
GeneJET, TransformAid and FastDigest are Fermentas trademarks.
Bacto Tryptone and Bacto Yeast Extract are registered trademarks of Difco Laboratories
GmbH.
PRODUCT USE LIMITATION.
This product is developed, designed and sold exclusively for research purposes and in vitro use only. The product was
not tested for use in diagnostics or for drug development, nor is it suitable for administration to humans or animals.
(1) Revised 02.05.2006
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