cloning large insert in pGEM-T easy vector - Does TA cloning work for big inserts? (Nov/28/2009 )
I am trying to clone a 3.8 kb insert into the pGEM-T easy vector, which is 3 kb big. It is just not working. Is it because the inset is bigger than the vector backbone? How can I circumvent this problem?
lotus on Nov 29 2009, 01:23 AM said:
No, it's not the problem. I have cloned a 5kb fragment using this vector and it's very convenient. You should show how you did that to figure out which step was problematic. Normally with this vector I perform PCR (with the Taq is able to make a 3'-A overhangs)-> purify by gel extraction (or sometime using the PCR cleaning kit if you have a sole product) -> ligate to pGEM-T by the molar ratio of vector : insert of 1:3 or 1:5 by incubating at RT for 1 hour or at 16 degree (if this is the hard case) -> transformation.
Beside checking the concentration of ligation components, activity of the Kit ligase and the condition of quick ligation buffer (how many times it was thawed-frozen, you can use the control insert in the Kit for positive control) maybe you should check whether your Taq has this activity or not. Otherwise you should perform additional step creating 3'-A overhangs.
3.8kb is not very big I cloned 5-6 kb to pGEM-T easy without a problem. I usually use 1:10 backbone to insert ratio, and I use only 25ng of pGEM-T. and if the ligation is problematic I do it at 4C for overnight, or most of the cases even longer (up to 1 week)