what's the best way to clone PCR products?

Kevin Morano ez005528 at rocky.ucdavis.edu
Thu Sep 22 13:25:36 EST 1994


Regarding PCR cloning:

1) Engineer restriction sites into the middle of your primers, leaving 
5-8 bp flanking on each side.

2) Geneclean/gel purify 5-10 ug of PCR band.

3) Digest cleaned PCR product AND host vector at least overnight. Pulse 
with fresh enzyme in the morning for 2 hours. Gel purify again.

4) Ligate overnight at 14 degrees C. Transform half, plate all.

THis method usually works well for us. Using CsCl pure vector frequently 
increases cloning percentages but miniprep DNA works well too. It is 
essential to start with a lot of DNA as you go through two gel 
purifications and the digestion efficiency on the PCR product is usually low.
-------------------------------------------

Kevin A. Morano
Internet:kamorano at ucdavis.edu    
Bitnet:kamorano at ucdavis   
Section of Microbiology, University of California, Davis



More information about the Methods mailing list