I am trying to make a site directed mutagenesis. For this, I have two set of primers (Anealling temp=55) designed which one of them produce an 600 bp amplicon and the other 1100 bp amplicon. I could produce them from cDNA. Now i want to sew these two amplicons from the PCR products that i have gained, however, the result I gain contains different size of amplicons smaller than the one which is expected (1700bp).
Your amplicons from first round of PCR, 600- and 1100-bp should have an overlap region to splice them together. This region should be unique and should have an annealing temperature equal to or higher than the primer pair you use in the second round of PCR. If these conditions are not satisfied, that may results in non-specific amplicons. Other recommendation would be to use a gel-purified PCR products from the first round of PCR as a template.
All the best!
Hi Solmaz,
You should design complementary oligonucleotide primers of about 30 nucleotide length with the point mutation in the middle of primers. Two sets of PCR amplification with an outer primer & a mutation primer will yield two amplicons in the first round of PCR (600 bp and 1100 bp respectively. Now set up a second round of PCR with EQUIMOLAR concentration of first round amplicons (gel-eluted) with outer extension primers, that will result in full-length amplicons with the desired point mutation.
Hope it helps.
Best!
Solmaz,
I have seen this problem before. it is most likely unspecific amplicons. There are a few remedies. I assume you used high fidelity polymerases and temperature gradient PCRs to amplify.
Change the PCR conditions, in regard of suffers, or even change the polymerases used. alternatively, try a seamless joining method such as the GIBSON reaction, SLICE, SLIC, etc.
good luck
P
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