PCR product - (May/19/2011 )
Can someone explain to me, why this happen???
This is my PCR products (from cDNA) using seed from tomato. Lane 1-6 are cDNA synthesized from RNA without any DNA removal while lane 7-10 are cDNA synthesized from RNA treated with DNase removal. Line 1 using 1st primer (with intron, PCR product= 536bp and without intron, PCR product=346bp). lane 3 using primer from the intron sequence (312bp). Lane 4 is housekeeping gene (UBQ=302bp). Lane 5 and 6 are other housekeeping genes. Same goes to the lane 7-10 except using cDNA with DNAse removal.
So, why did I got PCR product (lane 1) where the intron is still there and no PCR product on lane 7 after DNA removal?????
Do you understand that cDNA does not contain introns?
I do understand that. The problem is that, after DNase removal, I should get the band (on lane 7) with 346bp (without intron). But, I didnt get the band
Perhaps I'm being very silly, but I thought that there was that band in lane 7?
In lane 1 you (might) expect 2 bands - the band from the cDNA and the band from any genomic DNA still there. cDNA band without intron; gDNA band with intron.
In lane 7 you expect just one band - the cDNA band (no intron).
To me that's what your picture shows, but maybe I'm misreading it (what is the fuzzy band at the bottom?).
I might be misreading as I'm not sure what the ladder is and haven't looked at the other lanes except those 2.
the below bands are onle primer dimers. So, there's only 1 band in lane 1 (536bp, with intron) and no band on lane 7 (which I expected to get a band at 346bp). I used hyperladder I where the lowest band is 200bp.
OK, I see. In that case, the band is missing from both lane 1 and lane 7.
Do the other lanes show what you expect?
Did you check the RNA for degradation, for example by running it on a gel? Have you performed cDNA synthesis before without any trouble?