Hi,
I'm trying to amplify a DNA fragment using primers with Tm: 79.3℃ and 63.4℃. I have tried T gradients between 54-68℃. There's always a loylt of unspecific bands. Ithink that a touchdown PCR could work fine. What do you think?
Could this program be fine?:
95℃ 5'
10 times:
94℃ 1'
72℃ 1'
-0.5℃/cycle
72℃ 1'
29 times:
94℃ 1'
58℃ 1'
Sorry, the message follows:
72℃ 1'
Close loop
72℃ 10'
Thanks in advance,
Cecilia
The primer Tm is very high indeed.
If the high Tm comes from adding terminal sequences to gene-specific primers, try two rounds of PCR. In the first one, use only the gene-specific primers with no added sequences (Tm ~50-60oC) for ~16 cycles. Then use a 1/100 of this PCR product as a template for a second PCR which adds the tags/restriction sites/etc.
If that fails, I can warmly recommend trying to play with Mg (go lower if possible), DMSO (add to decrease non specific priming) and betaine (magical ingredient, honestly.) rather than annealing temperature alone.
Other suggestions: you do not mention the length of the amplicon or your template, but if it is short-ish, try to find restriction enzymes that do not cut it, and pre-digest the template DNA to reduce the probability of off-target amplification?
Good luck!
Hi,
You can try decrease MgCl2 concentration as much as you can. High MgCl2 concentrations promote losts of inespecific amplification.
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