Protocol Online logo
Top : Forum Archives: : Molecular Biology

RT-PCR - low mol.wt band (Nov/14/2008 )

Hi all,
I have designed the gene specific primers using the cDNA sequence of the gene.(1413bp) Then, I have put PCR using genomic DNA as template. I got 3kb band. then I thought, may be due to the presence of intron might have amplified high mol.wt band(3kb).
Then I put RT-PCR, expecting to get 1413bp band. but I got 250bp band (low mol.wt band). how should I analyse my result and how should I go ahead.

tons of thanx to all of U


if you are using random primers for RT reaction, that may give you mostly small fragments. I suggest also using oligodt primer for RT.


Hi cell counter,
thank U for the reply
I have used oligodt primer for synthesizing first strand cDNA and for the second strand synthesis I used the gene specific primer