Ligating small fragments into large vectors.

eb86336 at inet.d48.lilly.com eb86336 at inet.d48.lilly.com
Mon Sep 12 19:33:53 EST 1994


The answer to your question about UV light is NO.  Nicking can happen if you
expose your DNA to UV light for an extended period of time, but under normal
circumstances, this does not happen at a frequency worth being concerned about.

It sounds to me like your problem is that the 5 nt extension on your PCR primer
is not really long enough. If you look in the 1993/94 NEB catalog p. 181, you
will find that SalI which has 4 nt upstream of it has only a 10% cleavage
efficiency after 2 hr, and only 75% after 20 hr.  Therefore, the probable
answer as to why you are not getting any clones is that you are not really
cutting your 400 bp PCR product with SalI.

I would suggest that you purchase the pCR-Script kit from Stratagene for
cloning your PCR product directly, as this has been the best and most reliable
kit that we have used for direct cloning of PCR products.  You can then
subclone your sequenced PCR product on a SalI fragment into your vector of
choice.

I hope this helps.

Mike Menke
Eli Lilly and Company



More information about the Methods mailing list