Hello I am trying to amplify a plasmid DNA using a forward primer of 24 base pairs and reverse of 51 base pairs. In the reverse Primer among 51bp, 24 is hybridizing sequence and remaining 27bp is additional sequence that I wanna add to my target product (700bp).
My question is, Does it possible to add this 27bp additional sequence by PCR and If so then what can be the probable condition?
Hi,
Tm of 82 and 72 degrees are quite high knowing that the recommended elongation/extension temperature for most DNA polymerases is 72 degrees. Primers with melting temperatures above 65 degrees have a tendency for secondary structures. what is the GC content of your primers? you can reduce the hybridizing sequence of both primers to 18 nucleotides.
As Stefanno said, use only the annealing part for the Ta calculation, because that's, what matters to you, not the Tm. Look up for some polymerase, that is capable of two-step cycling.
Hi, I usually run 5 cycles with the lower annealing temperature (in your case corresponding to the Tm of the 24 bases of your rev primer), then I continue with a 2-step protocol (denaturation, e.g. 98°C––annealing/elongation, 72°C) for the remaining cycles. Good luck!
Hello,
you can also add DMSO (dimethylsulphoxide) into the reaction (usually total amount 5-10%), it also lowers the annealing temperatures. We are using it to allow running amplification of two products (annealing 60 and 68°C) at one PCR profile. It can be used in first as well as in seguencing reaction.
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