CloneJET PCR Cloning KitCloneJET PCR Cloning Kit is a positive selection system for efficient cloning of PCR products generated with any thermostable DNA polymerase. |
Features
The CloneJET PCR Cloning Kit contains a novel, ready-to-use positive selection cloning vector pJET1.2pJET1.2/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 pJET1.2pJET1.2/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.
- 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
For convenience in mapping and manipulation of the insert, the pJET1.2pJET1.2/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.
Note: Prior to electroporation, always column-purify the ligation mixture using e.g. GeneJET PCR Purification Kit #K0701 or chloroform to extract it. Electroporation is inhibited by the presence of proteins and salts in the mixture.
- 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
- pJET1.2pJET1.2/blunt Cloning Vector
- T4 DNA Ligase
- 2X Reaction Buffer
- DNA Blunting Enzyme
- pJET1.2pJET1.2 Forward Sequencing Primer (5’-CGACTCACTATAGGGAGAGCGGC-3’)
- pJET1.2pJET1.2 Reverse Sequencing Primer (5’-AAGAACATCGATTTTCCATGGCAG-3’)
- Control PCR ProductpJET1.2
- Water, nuclease-free
- Detailed Protocol
Hazardous | No |
---|---|
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. |
Storage Condition | -20ºC |
- 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
- pJET1.2pJET1.2/blunt Cloning Vector
- T4 DNA Ligase
- 2X Reaction Buffer
- DNA Blunting Enzyme
- pJET1.2pJET1.2 Forward Sequencing Primer (5’-CGACTCACTATAGGGAGAGCGGC-3’)
- pJET1.2pJET1.2 Reverse Sequencing Primer (5’-AAGAACATCGATTTTCCATGGCAG-3’)
- Control PCR ProductpJET1.2
- Water, nuclease-free
- Detailed Protocol
Hazardous | No |
---|---|
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. |
Storage Condition | -20ºC |
Supporting data
Procedure | Blunt PCR product | 3'-dA talled PCR product |
---|---|---|
Blunting | -- | 5 min |
Ligation | 5 min | 5 min |
Total time | 5 min | 10 min |
Table 1. CloneJET Cloning Procedure
pJET1.2/blunt vector map
Figure 1. pJET1.2/blunt vector map. pJET1.2/blunt cloning vector map. Click here for pJET1.2 sequence. |
Efficiency of different blunt-end PCR cloning systems with positive selection
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 1 x 107 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 1 x 107 transformants per µg supercoiled DNA. |
References
B. K. Michelsen, Transformation of Escherichia coli increases 260-fold upon inactivation of T4 DNA ligase. Anal. Biochem. 225, 172-174 (1995).
Resources
MSDS & Certificates CloneJET PCR Cloning Kit - MSDS Protocols and Product Manuals | Product Guides and Selectors Technical Resources pJET 1.2 Vector Map |