PCR primer with no extra DNA end (View forum version)



snpsnooper

Posted 27 September 2011 - 05:21 AM

I ordered a reverse primer that goes 5'-TCTAGA-xxxxxxxxxxxx. Forgot to add extra bases, will the Xba1 enzyme cut a pcr product amplified with this primer? Thank you for any feedbacks.

allynspear

Posted 27 September 2011 - 06:16 AM

New England Biolabs has excellent technical information on restriction enzymes cutting close to the end of DNA fragments. Here is a link:

http://www.neb.com/n...nucleotides.asp

XbaI does cut at much lower levels with 0 or 1 bases by the site, but it may still work. Alternatively, you could clone the PCR product into a T-Tailed vector like pGEM-T easy and then digest your fragment out of the plasmid.

Best of Luck.

snpsnooper

Posted 27 September 2011 - 06:28 AM

Thank you. I'll give it a whirl anyway. If it does not work then I'll just assume it is a blunt end (I used phusion pol.) and put it inside pbluscript or something and then cut it out of there later on.

phage434

Posted 27 September 2011 - 03:49 PM

Highly unlikely to cut.

snpsnooper

Posted 02 October 2011 - 02:40 AM

Highly unlikely to cut.


I didnt work. So I think you are right. Fallback option now: assuming that the Xba1 site is still blunted.

phage434

Posted 02 October 2011 - 06:25 AM

The blunt end will depend on the type of pcr enzyme you have used. If it contains Taq, a substantial number of the product fragments will have a 3' A overhang, which may inhibit blunt cloning. In any case, you will need to phosphorylate either the primers (before pcr) or the fragment (after pcr) to be able to clone, since the primer has no 5' phosphate.
You could consider TA cloning if you are using a Taq based pcr enzyme.