Hello. I am currently tagging a gene of my interest using the auxotrophic marker. The tagging primers which I am using for the process have a long stretch of A's (27) in their sequence due to which I am finding it difficult to tag the gene. Can anyone suggest me a way or some changes in reaction conditions? I am using Thermo Taq Polymerase for the same and the protocol provided on the Thermo site. Thank you.
Satyendra Mondal
Dear Rudrakshi Sharma
You can follow a few ways to troubleshoot your problem.
- Prepare a longer primer which possess an AT-rich region. AT-rich primers have lower melting point, so to adjust the melting point of two primers, the AT-rich primer length should be longer.
- Run a temperature gradient PCR to standardize the annealing temperature.
- Set the annealing step time a bit longer (30-40s) to have efficient annealing.
- If these don't help, choose a different restriction site containing higher GC-content.
Best wishes.
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Rudrakshi Sharma
If I have to use the same primer then what can I do for trouble shooting. I have already tried the temperature gradient, it didn't work plus I am following the protocol given in Janke etal, 2004.
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