3 replies to this topic

[greenlion25]

Hi all,

I'm trying to clone a 5 kb insert into pGem and have, thus far, had no luck.  I PCR amplify (using HF-Phusion) and then gel purify, extract from there using a Macherey-Nagel Spin Kit, A-tail my product, and then ligate into pGEM using NEB's T4 Ligase.  I'm not sure if the problem is with the A-tailing or with the ligation itself.  What concentration of PCR product should I use in the A-tailing reaction - ie does it matter that my insert is big (using Taq, Buffer, and MgCl2, in addtion to dATPs)?  All components are fresh and that isn't the problem.  I've also read elsewhere that a low insert:vector ratio (ie 1:1) should be used for large inserts.  Anyone have an opinion on this.

Thanks!

[phage434]

Is your pGEM cut somehow to leave single base T overhangs?  Or, perhaps better, how is your pGEM vector prepared for ligation?

[greenlion25]

phage434, on Oct 29 2009, 09:10 PM, said:

Is your pGEM cut somehow to leave single base T overhangs?  Or, perhaps better, how is your pGEM vector prepared for ligation?

pGEM is cut, linearlized, and de-phos.   same batch of pGEM is working fine for others in the lab.

[phage434]

pGEM, when cut, does not have T overhangs.  You may be referring to the Promega pGEM-T product, a pre-cut plasmid which has been cut with EcoRV and had T overhangs added.  Are the people who are having success using TA cloning, or are they doing a normal cohesive end cloning reaction?