No amplification during RT-PCRcDNA synthesis problem?
Posted 16 March 2013 - 04:24 AM
Is the larger amplicon size a problem and shall I expect that because of some problems during my RT reaction, larger RNAs have not completely reverse transcribed? Are there any modifications which can be made during cDNA synthesis to get rid of this problem?
Posted 16 March 2013 - 08:08 AM
If you are able to amplify additional genes from your recently produced cDNA, I would think that it is your current PCR method. Try new dNTP's, new buffer, maybe a new aliquot of your polymerase to see what happens.
Posted 16 March 2013 - 08:54 AM
It was less than a week old. If RNA is a problem, shouldn't amplification of other genes also get hampered? Yes, with previous cDNA current primers are giving brilliant amplification. No, I don't expect downregulation of the gene in the present cDNA. With the same set of PCR reagents previous cDNA is amplifying; also amplifying is the other set of genes with the present cDNA. So I don't blame the PCR reagents.
Posted 16 March 2013 - 10:17 AM
Posted 17 March 2013 - 05:51 PM
if your 200bp amplicon is being amplified that means either you have problem with primers for 2.5kb fragment or your RNA is degraded.