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PCR-TRAP Cloning System

$370.00
SKU:
P404
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Product Description

For positive-selection cloning of PCR products through blunt-end ligation. Allows direct cloning of PCR products without purification.

 

GenHunter’s PCR-TRAP® Cloning System is the most efficient method for cloning PCR products by blunt-end ligation. This system uses a third generation cloning vector that features positive selection for cloning PCR products (see figures next page).  Only recombinant plasmids confer antibiotic resistance, making PCR-TRAP® extremely efficient.  There is no need for any post-PCR manipulation before cloning, since a significant fraction of PCR products do not contain the 3’ overhanging A (Clark, 1988, Nucleic Acids Res. 16:9677).

The mechanism of this unique cloning system involves the phage Lambda repressor gene, cI, which has been incorporated into the PCR-TRAP® Vector.  When transcribed, the gene codes for a repressor protein which binds to the Lambda right operators Or1 to Or3 of the cro gene, turning off the promoter which drives the TetR gene on the plasmid.  Therefore, cloning the PCR product directly into the cI gene leads to the inactivation of the repressor gene and thus the expression of the TetR gene.  So the PCR-TRAP® Cloning System is indeed a cloning TRAP!  

The cloned PCR product can be quickly and easily retrieved by colony-PCR for the purpose of insert confirmation or probe generation using primers which flank the cloning site (provided in the kit).  If cDNA amplified by an RNAimage® or RNAspectra® Kit is cloned into a PCR-TRAP® System, the insert can also be excised by HindIII digestion.  The flanking primers also allow the cloned PCR product to be readily sequenced.

The kit contains GH Competent cells as well as a positive control, the lacZ gene, which after PCR and cloning will confer blue color upon X-gal staining.  This kit is also ideal for conducting reverse Northern blot screening for positive clones during differential display (Ref. 2 and 3).

  PCR-TRAP Cloning System (for 30 clonings)
 

   1.   Insert-ready PCR-TRAP® Cloning Vector

           (150 ng/µL)

60 µL
   2.   T4 DNA Ligase (200 units/µL) 30 µL
   3.   10X Ligase buffer 70 µL
   4.   dH2O 1.2 mL
   5.   Colony Lysis Buffer (5 vials) 5 mL
   6.   10X PCR Buffer 500 µL
   7.   Lgh Primer (2 µM) 200 µL
   8.   Rgh Primer (2 µM) 200 µL
   9.   dNTP (250 µM) 200 µL
   10. pUC18 control template 10 µL
   11. Llac Primer 20 µL
   12. Rlac Primer 20 µL
   13. Tetracycline 1 mL
   14. Competent cells 3 mL

References:

  1. Liang, P.:  Analysis of messenger RNA by Differential Display. A laboratory guide to RNA. 1996, pp223-236.
  2. Vogeli-Lange, R. et al.:  Rapid selection and classification of positive clones generated by mRNA differential display. Nucleic Acid Res. 1996, 24:1385-1386.
  3. Zhang, H. et al.: Differential screening of gene expression difference enriched by differential display. Nucleic Acid Res.  1996, 24:2454-2455.
  PCR-TRAP Cloning System (for 30 clonings)
 

   1.   Insert-ready PCR-TRAP® Cloning Vector

           (150 ng/µL)

60 µL
   2.   T4 DNA Ligase (200 units/µL) 30 µL
   3.   10X Ligase buffer 70 µL
   4.   dH2O 1.2 mL
   5.   Colony Lysis Buffer (5 vials) 5 mL
   6.   10X PCR Buffer 500 µL
   7.   Lgh Primer (2 µM) 200 µL
   8.   Rgh Primer (2 µM) 200 µL
   9.   dNTP (250 µM) 200 µL
   10. pUC18 control template 10 µL
   11. Llac Primer 20 µL
   12. Rlac Primer 20 µL
   13. Tetracycline 1 mL
   14. Competent cells 3 mL

Documents

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PCR-TRAP Kit (for 30 clonings)   - Cat. No. P404