5 replies to this topic

[seed]

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?????

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Edited by seed, 19 May 2011 - 07:19 AM.

[Lapsang]

Do you understand that cDNA does not contain introns?

[seed]

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

[Lapsang]

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.

Edited by Lapsang, 20 May 2011 - 05:07 AM.

[seed]

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.

[Lapsang]

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?