Hi,
I would like to amplify a piece of cDNA (prepared from mutant ES cells RNA) coding for a gene after Cre recombination (so the gene has one exon less).
I designed a pair of primers with NdeI or BamHI ends for forward and reverse primers respectively.
However, after PCR, the PCR amplicon obtained is below 100bp and I was expecting a 288bp band.
I repeated the experiment with a gradient PCR to determine the best annealing temperature. Despite those efforts, I still do not get the expected size product.
Do you have any clue why it is not working?
Thank you
You would need to sequence your product and see what it is.
I would also try a nested PCR or a PCR without the restriction enzyme site addition, followed by the restriction enzyme ends PCR.
How long are your primers?
Thank you Isaac for your suggestions.
Actually I tried to sequence it, but it did not work, the sequencing has not been optimal.
The primers are both 20bp.
My guess is you are having issues with dimerization. try a nested PCR
As your primers contain NdeI or BamHI ends, I would recommend increasing the primer lengths to 26bp.
Hi Paul,
For me, If I got a non-specific band from PCR, the first thing was check the primer: with higher Tm or less dimer. The website IDT (integrated DNA technologies) is very useful and convenient for primer design. Then I will check the DNA Polymerases, if you want to make a construct, I suggests that you can use the "Phusion® High-Fidelity DNA Polymerase" from NEB. Anyway, if your target fragments contains high AT or GC content, you may change the component of reaction buffer, such as lower the magnesium or add DMSO.
Good luck!
Yimin
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