I am a synthetic/medicinal chemist. We are currently doing a DNA encoded library of small molecules, we plan to label each small molecule with about 40 bp ssDNA. Now I can add few bp for primers in each side as the compliment for two primers. With this understanding I ordered a DNA of 80 bp with 20 primer binding site at each side. We did the PCR first and saw products only at 51-52 0C and then did qPCR with the same condition, ie; 52 0C. Unfortunately we only got the Ct values in range of 30-35 and not determined in low concentration. Reactions concentration was serially diluted from 500 uM ssDNA template.
Now the questions are,
Is the Tm value 52 is okay or we need to redesign the primers to make that in range of 55-65 ?
What other factors may be used to better the Ct values ?
Thanks every one
Thank you Jose, We did not do the melting curve analysis. We will do that and see what is happening. Also the DNA template concentration we used were from 500 uM to 50, 5 and 0.5 uM. We got a Ct of 30 when we used 500 uM. I was skeptical about the Tm 52 and did not do anymore experiments till. But sure I will do some tomorrow and will discuss the outcome then
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