CloneJET™ PCR Cloning Kit

- Store at -20°C

Catalog#	Size, concentration	Certificate of Analysis	MSDS

K1231	for 20 react.	K1231
K1232	for 40 react.	K1232

Product information

Features

Fast – PCR cloning in only 5 minutes.
Highest efficiency – >99% of positive clones.
No cloning background – positive selection vector.
Versatile – ideal for blunt-end or sticky-end cloning.
Economical – no expensive blue/white screening.

Applications

Cloning of blunt-end or 3'-dA tailed PCR products up to 10 kb.
Cloning of DNA fragments generated by restriction enzymes.
Sequencing of cloned DNA.
in vitro and in vivo transcription of cloned inserts from the T7 promoter.

Description

The CloneJET™ PCR Cloning Kit is an advanced positive selection system for high efficiency cloning of PCR products generated with any thermostable DNA polymerase. Additionally, any other blunt or sticky-end DNA fragment can be cloned. The kit is ideal for phosphorylated or non-phosphorylated DNA fragments. Ligation into the included positive selection vector takes only 5 minutes and yields more than 99% recombinant clones. Blunt-ended PCR products generated with a proofreading enzyme (e.g., Pfu polymerase) are ligated directly into the cloning vector. PCR products generated either with non-proofreading DNA polymerases (e.g., Taq polymerase) or mixtures of DNA polymerases are blunted prior to ligation in 5 minutes with the thermostable DNA Blunting Enzyme provided with the kit. All common laboratory E.coli strains can be directly transformed with the ligation product.

The kit contains a novel, ready-to-use positive selection cloning vector pJET1.2/blunt. The vector contains a lethal restriction enzyme gene that is disrupted by ligation of a DNA insert into the cloning site. As a result, only bacterial cells with recombinant plasmids are able to form colonies. Recircularized pJET1.2/blunt vector molecules lacking an insert express a lethal restriction enzyme which kills the host E.coli cell after transformation. This positive selection drastically accelerates the process of colony screening and eliminates additional costs required for blue/white selection.

For convenience in mapping and manipulation of the insert, the pJET1.2/blunt cloning vector multiple cloning site contains two BglII recognition sequences that flank the insertion site. In addition, the vector contains a T7 promoter for in vitro and in vivo transcription as well as sequencing of the insert.

Quality Control

The kit is functionally tested by cloning a control 3'-dA tailed PCR product. Typical yield of recombinant clones is >99%. The cloning vector is tested in a self-ligation experiment for the absence of cloning background. The pJET1.2 primers are tested for specific and efficient sequencing.

Components

pJET1.2/blunt Cloning Vector (pJET1.2 DNA sequence in txt format)
T4 DNA Ligase
2X Reaction Buffer
DNA Blunting Enzyme
pJET1.2 Forward Sequencing Primer (5’-CGACTCACTATAGGGAGAGCGGC-3’)
pJET1.2 Reverse Sequencing Primer (5’-AAGAACATCGATTTTCCATGGCAG-3’)
Control PCR Product (sequence of control PCR product in txt format)
Water, nuclease-free
Detailed Protocol

Note

Prior to electroporation always column-purify the ligation mixture using e.g. GeneJET^TM PCR Purification Kit, #K0701 or chloroform extract it. Electroporation is inhibited by the presence of proteins and salts in the mixture.

Table 1. CloneJET™ Cloning Procedure.

Figure 1. pJET1.2/blunt vector map.
pJET1.2/blunt cloning vector map (pdf, 188 KB). pJET1.2 DNA sequence (*.txt file).

Figure 2. Cloning of blunt-end PCR product: efficiency of different blunt-end PCR cloning systems with positive selection.
A 976 bp blunt-end PCR product, generated with Pfu DNA Polymerase, was directly ligated into different positive selection blunt-end PCR cloning vectors according to suppliers protocols. Ligation mixtures (2 µl) were used to transform E.coli DH10B cells. Transformation efficiency of competent cells was 1x10⁷ transformants per µg supercoiled DNA.

Efficiency of different PCR cloning strategies for 3'-dA tailed PCR product

Figure 3. Cloning of 3’-dA tailed PCR product: efficiency of different PCR cloning strategies.
A 976 bp 3’-dA tailed PCR product, generated with Taq DNA Polymerase, was ligated into pJET1.2/blunt and into different TA cloning vectors according to suppliers protocols. Ligation mixtures (2 µl) were used to transform E.coli DH10B cells. Transformation efficiency of competent cells was 1x10⁷ transformants per µg supercoiled DNA.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

References

Michelsen, B.K., Anal. Biochem., 225,172, 1995.

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