I already did my RT, but now I want to do a PCR to replicate all my cDNA, is it possible to do a PCR with the hexamer random Primer as well? I do not find any KIT for PCR that is not with gen specific primers.
Hi,
If you have double-stranded DNA (you made second strand synthesis after RT or used Template switch approach during RT, etc.) and want to amplify the whole cDNA you may use couple of approaches: if you OK with fragmenting your cDNA, you can use Nextera (usual/XT) kit from Illumina. Tn5 transposase will cut your cDNA with inserting adapters for further PCR using N5XX and N7XX Illumina primers. The size distribution will be 200-600 bp roughly which is fine for NGS, but inappropriate for gene-specific qPCR, if the primers cover longer sequence. If you do not want to fragment the cDNA that much, you may try a Covaris&Clontech (cat 634900) library prep (they first do the ultrasound fragmentation on Covaris, but you may try to skip it or sonicate with less time/power to increase the length of the particles), continuing with polishing the ends, adapter attaching and PCR.
Best,
Michail
The approaches discussed by Ali and Michail are good to follow. However, if you still wish to amplify the total cDNA, you have to use hexamers tagged to known but unrelated sequences, which can serve as primer binding sites in the next step. in the next step, you can use primers binding to the the tag sequence for PCR. This is known as SISPA and you can go through the attached paper for details of the procedure.
best of luck.
SD
Article Use of Sequence-Independent, Single-Primer-Amplification (SI...
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