I have been trying to amplify a puromycin insert from a plasmid from Addgene. I have tried Hot-Start reaction, Pfu turbo polymerase- the later has been a little bit better. Still, not getting enough product for downstream ligation & cloning though. Primer Tm values are 50 and 60.
Suggestions?
Did you try lower annealing temperatures ?? Lower temeperatures may increase your specific product, but can also give you non specific bands. So be careful as to how low you go. Gradient pcrs help to decide that(45-55 since your tm is 50). Another issue could be the polymerase. Try a different high fidelity polymerase.good luck.
why are you doing TAIL-PCR on an Addgene plasmid? Do you mean thermal asymmetric interlaced PCR? Do you not know the sequence that you're trying to amplify? It would be much easier to obtain a plasmid with a known sequence and design the appropriate primers.
TAIL-PCR is Thermal asymmetric interlaced polymerase chain reaction and generally used for determination of the insertion site of a know element (for example transposon). Do you mean PCR using primers with tails for subcloning? Try Phusion II or PrimeStar proofreading polymerase, these are potent enzymes.
Thank you all for helpful suggestions. I have attempted some of the suggestions above, but not much luck yet. I will now try your suggestion, Markus, of linearizing the plasmid before amplifying the insert.
The insert from the Addgene plasmid I wish to isolate for subcloning is flanked by loxP sites- highly AT rich. I think that's part of the poor yield I get- perhaps the primers are forming secondary structures etc..??
I mentioned TAIL-PCR because I had better luck in genomic PCRs where target sequence had AT-rich segments- by doing second round only of the TAIL-PCR, I was able to successfully amplify the area of interest. So I wondered if the same would be applicable for this plasmid as well.
Any additional suggestions/feedback on my approach?
Thank you all!!
@Srinivasan- I have tried gradient PCR- with negative results. In turns, I also tried lowering the annealing temperature, the extension temperature..also without positive outcome.
I have tried HotStar master mix previously, that did not work at all. Now trying with Pfu turbo polymerase, somewhat better. But as I mentioned above, not getting enough yield for ligation.
I would recommed that you look at the method for TAIL PCR described in this paper: Liu YG, Mitsukawa N, Oosumi T, Whittier RF: Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR.
Plant J 1995, 8:457-463
It is a three step PCR reaction where you get higher target specificity for each nested PCR. We used an adapted version of this method to succesfully identify more than 120 transposable inserts using just normal Taq polymerase (see BMC Genomics. 2011 Mar 20;12:155. doi: 10.1186/1471-2164-12-155).
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