I have been struggling for over 2 months with a PCR. I am trying to check if a gene is expressed in the intestinal tissue of zebrafish. Following total RNA extraction with Trizol, I do a DNAse treatment and then a reverse transcriptase reaction to obtain cDNA. The problem that is continuously occurring is that sometimes I can see the expression and at other times I don't. The positive control (a housekeeping gene called ODC1) is consistent however. I am struggling to understand the reason for the difference in results, especially since I have used the same pool of cDNA for all these reactions. I have not changed any settings on the PCR machine either. The only different parameter thus are the primers. Anyone else having this experience? Thank you.
it is likely that you are not using the optimal tm for your primers...I would advice you to make a gradient temperature to check in which tm you'll obtain a stronger amplification during the pcr...
You can check your primer pair by running it in 4% agarose gel which will give u the status of primer. Primer degradation happens when the primer undergoes multiple freeze and thaw cycle. Also, check different dilution of cDNA ( 1:10 and 1:15).
David, I've tried a temperature gradient and that gave me an inconclusive result. I got a good range for the controls but got no results with the gene of interest. This again goes back to the original problem where my pool of cDNA works at times and doesn't t other times.
Thank you Vivek. Can you please explain a little more in detail? How does a 4% gel give the status of a primer?
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