The annealing temperature of the primer is very low (35 degrees centigrade). What should be the measures? Should I redesign my primers?
Low GC contents and/or short length of primers results in very low annealing temperature. At this low annealing temperature poorly designed primers makes hairpins and dimers. I think you can try to optimize annealing temperature using gradient PCR , if it does not work then you may need to design new primers. I can check the quality of primer design if you let me know the sequence before ordering. You can use following link to design primers.
http://www.yeastgenome.org/cgi-bin/web-primer
Can you please give us more details (probably the primer sequence if you want), at the moment its a bit like reading the crystal ball. And yes, 35°C for annealing is most likely too low.
Dear Ria can you be more clear about the primers or upload the sequences. I agree that 35°C is indeed very low. Anyway here is a website that can help you test and evaluate your primers: http://www.basic.northwestern.edu/biotools/oligocalc.html
Pay attention to things like GC content, ending the sequence with a G or a C, tm of each primer (of a pair) must be close together (or PCR will be very inefficient or not possible). Good luck.
35°C sounds excessively low, what is the length of your primers? The usual 18-20 bases? If they are too short, they might not be specific enough.
At the moment its a bit like reading the crystal ball. And yes i completely agree that 35°C for annealing is most likely too low. please Give more details.
35°C? At that temperature anything will hybridize. Send me the sequence of the fragment you want to amplify and I will give you the primers....
If you have a 3step PCR program, and increase the temp from the annealing step (35°) to 72° at the elongation step, your hybridized primer with the first bases that were added, will just separate from your template.
Yes 35°C is low. What is the length of your primer. Have you tried higher temperature for annealing. If not take a chance with 45°C. It might work. Good luck
Without increasing the Tm of the primers by redesigning with good GC ratio, increasing randomly the annealing temperature is not procedurally correct, U may be working beyond the Tm, finally yielding nothing......
The best is to redesign the primers.
I agree to redesign the primers. However, have you a contol sample to test them?
You can perform a gradient PCR from 35-55 to see if you can increase the temperatureof your reaction. I have worked succesfully with primers with 35-40oC.
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