This primer is supposed to amplify 122bp amplicon of cDNA... I wasn't able to get the desired band through conventional pcr so I tried qpcr for a single sample. Ct came out 27 and melt curve attached. But when I ran on gel it shows up below 100bp. My primer length is below 25 and tm of both is 64.. any advice will be appreciated if I should trust melt curve or the gel..
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I extracted RNA from PBMCs it has been consistent with my samples I always get low A260/230 VALUES <1.. I know it's guanidium contamination or maybe ethanol. But I have already made cDNA want to know if it will inhibit my qPCR ? Any one who has experienced this plz send advice. Also I used RNA only 1000ng for 20 ul reaction mix.
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I extracted RNA from PBMCs it has been consistent with my samples I always get low A260/230 VALUES <1.. I know it's guanidium contamination or maybe ethanol. But I have already made cDNA want to know if anyone has experienced this and their qPCR worked or not? Any one who has experienced this plz send advice. Also I used RNA only 1000ng for 20 ul reaction mix so it's very much diluted.
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