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CloneJET™ PCR Cloning Kit
The efficient system for fast cloning of PCR products

#K1231	for 20 reactions
#K1232	for 40 reactions
Related Documents (in pdf, ~200 KB): Detailed Protocol: #K1231, #K1232 MSDS (English) MSDS (English-USA) MSDS (German)
pJET1.2/blunt cloning vector map (pdf, 188 KB); pJET1.2 DNA sequence (*.txt file) Sequence of control PCR product (*.txt file)

 

Features

• Fast - 5 min cloning of PCR products.
• Highest efficiency - greater than 99% of positive clones.
• No cloning background - positive selection vector.
• Universal - suitable for cloning of blunt-end or sticky-end DNA fragments with or without 5'-phosphates.
• Economical - expensive blue/white screening is not required.
• Convenience of pJET1.2 cloning vector:
  • ready-to-use: linearized, blunt-end, contains 5'-phosphoryl groups
  • MCS designed for easy mapping and subcloning of a cloned insert
  • T7 promoter for in vitro transcription of the insert
  • Double BglII site flanking the cloning site for easy excision of insert.

Description
The CloneJET™ 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 other thermostable DNA polymerases. Additionally, any other DNA fragments, either blunt or sticky-end, can be successfully cloned using the kit. The cloning is fast and efficient. The ligation takes only 5 minutes and yields more than 99% of positive clones.
For efficient cloning of sticky-end DNA fragments, e.g. dA-overhangs on PCR products generated by Taq DNA Polymerase, a unique DNA Blunting Enzyme is provided with the kit. Blunting procedure takes only 5 min.
The kit features a novel positive selection cloning vector pJET1.2/blunt. The vector contains the lethal restriction enzyme gene which is disrupted by ligation of a DNA insert into the cloning site. As a result, only cells with recombinant plasmids are able to propagate.
For convenience in mapping and manipulations with the insert the pJET1.2/blunt cloning vector provides multiple cloning site with the double BglII site flanking the insertion site. In addition, the vector contains T7 promoter for in vitro transcription of the insert.

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

Procedure	Blunt PCR product	3'-dA tailed PCR product
Blunting	-	5 min
Ligation	5 min	5 min
Transformation of the cells prepared with the TransformAid™ Bacterial Transformation Kit	5 min	5 min
Plate	immediately	immediately
Total time:	10 min	15 min

Applications

• Cloning of blunt-end PCR products, generated with Pfu or other proofreading DNA polymerases.
• Cloning of 3'-dA tailed PCR products.
• Cloning of DNA fragments, generated by restriction enzymes.
• Sequencing of cloned DNA.
• In vitro transcription of cloned insert.

Quality Control
The kit is function-tested by the 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 of the Kit

• pJET1.2/blunt Cloning Vector
• T4 DNA Ligase
• 2X Reaction Buffer
• DNA Blunting Enzyme
• pJET1.2 Forward Sequencing Primer
• pJET1.2 Reverse Sequencing Primer
• Control PCR Product
• Water, nuclease-free
• Detailed Protocol (in pdf, ~160 KB):
  • #K1231, #K1232

Figure 2. Cloning of blunt-end PCR product: efficiency of different blunt-end PCR cloning systems with positive selection of recombinant clones.
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.

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.

Related Products

PyroStart™ Fast PCR Master Mix (2X) Pfu DNA Polymerases Taq DNA Polymerase: recombinant native High Fidelity PCR Enzyme Mix Long PCR Enzyme Mix dNTP Mixes dNTP Set TransformAid™ Bacterial Transformation Kit GeneJET™ Plasmid Miniprep Kit DNA Gel Extraction Kit FastDigest® Restriction Enzymes Ampicillin E.coli FastMedia™ pJET1.2 Sequencing Primers E.coli strains GM2163 (#M0099), JM107 (#M0109) can be provided free of charge upon request, with any purchase.

Updated balandžio 24, 2008 18:52