I am doing TA cloning using PTZ57R/T vector. Positive control is getting transformation, but my poly A tailed amplicon after ligation with TA vector is not getting transformed. Why is this?
I have carried ligation at 22C for 1hr, 16C for overnight and 4C for overnight all were carried for transformation and I didn't get a single colony. I have made sure that my buffer and ligase enzymes are good. I used concentration of 3:1 insert: vector ratio.
PolyA tailing:
after carrying PCR with taq polymerase. I have added 10mM dATPs to the same reaction mixture and carried polyA tailing is this a wrong method? Do I need to gel purify the amplicon and do polyA tailing?
Temp for polyA tailing is 95C for 10min and 72C for 20min.
try different insert: vector ratio like 1:7 and 1:5 and try keeping in 4 degree celsius for half an hour only after you have done ligation at 16 degree celsius and then transform the ligated product
We are using TA for direct cloning of PCR products made with Taq polymerase. Taq polymerase adds single 3' adenines to both sides of fragment, which is then ligated into vector with single T overhangs at 5'. So I am not sure why you are performing tailing reaction. Just try to amplify your fragment with Taq polymerase and ligate it straight into properly prepared vector.
May be you can try to run the ligation reaction on a gel and see if the ligation is going fine. If you have a good amount of DNA you might be able to see if you are getting circular plasmids in your ligation. Of course you should run a plasmid alongside your ligation reaction to check what size of bands you are getting with circular plasmid. Then if you see that you have circular plasmids but still not getting any colonies after transformation I would think that your cloned product might be toxic to the E coli and you might want to change the strain you are using for transformation. There are some strains available from different companies suggested to be used with toxic genes.
Also use cells which are ultracompetent or supercompetent. That might also help...
As to your question about PCR product. I would gel purify the product first and then do the polyA tailing.
Good luck with your cloning! Hope it works in the end!
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