I am using sybr green mastermix and my cDNA quantity is okay. Still not getting amplification curve both for expected gene and internal control (GAPDH)
You might consider the following options:
1. make new primer dilutions from the stocks
2. use/make new aliquots of cDNAs; maybe they have undergone too many freeze/thaw cycles
3. use a fresh aliquot of SYBR mastermix; maybe the one you are using underwent too many freeze/thaw cycles
4. make sure the cycling parameters are correct
Do others using the same instrument still get amplification since your problem started? Or do you use other assays on that instrument that are still working? Maybe the instrument is not heating/cooling properly (in which case nobody should get amplification with this instrument for any assay).
Thanks a lot.My problem has been solved.My cDNA was too much concentrated. Then I made a dilution of 1:5.now I am having the results. It’s a great lesson.
Honestly, "dilute your cDNA" is a lesson it seems far too many people need to learn the hard way. For the record, even 1/20 is usually fine for qPCR (effectively this just shifts all your data 2 cycles later than 1/5 dilution, which usually is fine even for low abundance transcripts). This also makes your cDNA last a lot longer.
Glad you solved it, but be sure to tell everyone you discuss this with that "dilute your cDNA" is the way to go.
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