My cDNA is Pure: 1.89 and quantity: 2525. Previously I used the same cDNA for a pair of primers and got a concentrated band in a 20ul PCR reaction mixture (10uM each working primer), but in the next several PCRs they are consecutively giving negative results, however, both cDNA and primers are tested positive? Is there any issue with PCR? I didn't changed the protocol.
was there a long time gap between the cDNA working and the first time that the cDNA did not work with the same primers. Have you run other primer sets recently on the cDNA and had positive results? Can you amplify genomic DNA with the failing primers to get a longer band but proving that the primer pair and reagent set do work?
Dear Ismail Zeb,
Some of the possible reasons for your problem may be of:
1. c-DNA stored for a long time or didn't store properly. Check once again the quality of cDNA synthesized by nanodrop and 0.8% agarose gel. (If it's Ok...)
2. Do check with housekeeping genes with the same c-DNA...
3. Make sure that the agarose gel and the running buffer are fresh and new. Because many lab are reusing the gel and running buffer for a long time. (You can see the running buffer by change in color from colorless to light blue one).
I have given only 3 possible reasons provided I hope you have autoclaved your tips, PCR tubes and other essentials before use.
And also make sure the primers are not degraded and milliQ water is without any DNase...
1. Check the activity of polymerase (sometimes after few freezing cycles the activity drops significantly), if the polymerase is good ...
2. Try to amplify a housekeeping gene with your cDNA. In the case of negative results, maybe your cDNA has been degraded
3. If you get the positive result with housekeeping gene, try diluting a new pair of primers
take fresh enzyme, make sure there is no contamination esp in PCR water.
Thank you all for your suggestions, i will concern all the suggested tips.
Previously I was facing the same problem, I resolved the issue by diluted concentrated DNA sample. As You mentioned the your concentration of DNA Sample is high You should make 10 picomol dilutions. Also check the nano drop value of primers sometime primers are contaminated.
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