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I have treated my RNA with sodium bisulfite so all my cytosine is converted into thymine. Then I used that RNA for my RT reaction. I have a cDNA template without Cs. Now I am trying to amplify that region with PCR.

For some reason I am not able to amplify the region. I designed my primer by Primer 3 plus and manually, but it is not working. The main reason is that I don't have GC pair in the primer.

Is there any other way to fix the problem?

My primer concentration is 200nm.

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  • $\begingroup$ 1) Are you sure all the cytosines in your RNA were converted to thymines? These assays are usually done to determine which bases are protected by binding to other macromolecules or to secondary structures in the RNA. 2) Even if you converted all Cs to Ts you would still have Gs that will pair with Cs when you make the first strand of cDNA — thus, saying there are no GC pairs can't be correct. 3) It might also help if you included the expected original (pre-bisulfite treatment) primer binding sequence. $\endgroup$
    – tyersome
    Dec 31 '19 at 22:27
  • $\begingroup$ yes, all the Cs converted into Ts, i amplified the very short region then i confirmed by sequencing. In the genome there are some Cs are methylated i want to target them. My question is within the primer I don't have GCs. My primer is not annealing properly i am getting only Primer-Dimer. $\endgroup$ Jan 1 '20 at 8:14
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Without the sequence information I can only give very general advice.

1) Pick a different location — primers generally work best when they have something close to 50% GC content.

2) Make sure the 3' ends of your primers aren't complementary.

There is detailed advice on how to design effective primers available online including:

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  • $\begingroup$ Somehow i managed to amplify the region of interest. I used HIFI-Kapa PCR mix. I kept $\endgroup$ Jan 12 '20 at 11:46

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