RT-PCR primer design for full length cDNA cloningRT-PCR cloning
Posted 22 August 2011 - 03:12 PM
I’m currently planning an RT-PCR in order to clone my gene of interest. I’ve settled on a 2-step RT-PCR protocol using a specific primer at the retro-transcription (RT) step.
I’m now trying to design primers, one specific primer for the RT step and another pair for the PCR step having restriction site in 5’.
I understood that the reverse primer for the PCR step must contain the stop codon (as well as the forward must contain Kozac sequence and start site). However I get a little confused with the specific primer I want to design for the RT step. Indeed, to me that first primer must be very close – if not identical – to the reverse primer of the PCR step.
So my questions is: can I use the same reverse primer for RT AND PCR steps.
Is it something usual? And if so I suppose nothing prevents me from having a primer for RT step that possess a non-complementary RE site in 5’? Any good advice on this or closely related are welcome.
Finally, and I think this is linked somehow, I would like to know why the only type of priming possible in a one-Step RT-PCR is using a specific primer; I don’t see why hexon priming or oligodT priming would impeed the PCR step of the reaction, or is it due to another concern…
Thanks a lot for your future answers, can’t stand waiting to discuss that
Posted 22 August 2011 - 05:48 PM
Oligo dT primers offer specificity toward polyA mRNA, but if the mRNA is too long, you may not get full length cDNA (the reaction could not reach the very 5 end of the mRNA). Gene specific primers offer even more specificity toward your gene, but may have the same concern as oligo dT primers.
If you do want to use a gene specific primer for the RT reaction, you can use the same primer as the reverse primer in subsequent PCR reaction. You can also design a GSP in the 3UTR for the RT reaction, then use use another reverse primer which is upstream of the GSP for the RT-PCR.
Posted 23 August 2011 - 06:16 AM
And I understand why hexamer priming is impossible in a one-Step RT-PCR, but eventually wouldn't oligodT priming be possible in this context where the tube is not opened from the beginning to the end of the reaction ?
Posted 23 August 2011 - 07:09 PM
Posted 24 August 2011 - 07:36 AM
Posted 24 August 2011 - 10:38 PM
Posted 29 August 2011 - 02:35 AM
however, i dont see the band after running the gel, although the gapdh band is very strong. also RNA is not degraded at all.
so i was wondering if it is possible because the cdna is not fully transcribed, but how come the gapdh still works.
could you guys help. thanks.