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RT-PCR stoped working!!! Same samples, same primers, same enzyme!


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#1 csmartinho

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Posted 02 August 2011 - 05:10 AM

Hello all,

I would appreciate if anybody could share a possible solution for the problem I'm having with RT-PCR.

I was doing semi-quantitative RT-PCR for a specific transcript that was not very abundant. Usually requiring between 35 and 40 cycles to get signal, independently of this it was working perfectly and we were able to a very good and reproducible amplification.

However, suddenly this SAME pcr, with the SAME primers and reagents started to fail. Instead of bands we get smears, which also appear in the blank, suggesting a contamination with degraded DNA (possibly). Even though this does not amplify anything from the cDNA, it amplifies perfectly genomic DNA!!! The cDNA is ok because I am able to amplify other transcripts. Since the fragment has 1kb we also tried RNAseH treatment but this did not help. I tried even to order a new stock of the same primers, but it didn't change anything. Finally we also tried to amplify old cDNA samples in which we managed to amplify successfully this transcrip before and now IT DOES NOT work!!!!!! I'm REALLY puzzled, does anybody has any hint on this problem?


Thanks a lot,
ClŠudia

#2 csmartinho

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Posted 04 August 2011 - 03:13 AM

I manage to make it work now once, when trying a different Taq, however it worked only for the usual Taq and in the next attemp stoped working. There's a lot of variability and a co-worker also could not do it. :(


Hello all,

I would appreciate if anybody could share a possible solution for the problem I'm having with RT-PCR.

I was doing semi-quantitative RT-PCR for a specific transcript that was not very abundant. Usually requiring between 35 and 40 cycles to get signal, independently of this it was working perfectly and we were able to a very good and reproducible amplification.

However, suddenly this SAME pcr, with the SAME primers and reagents started to fail. Instead of bands we get smears, which also appear in the blank, suggesting a contamination with degraded DNA (possibly). Even though this does not amplify anything from the cDNA, it amplifies perfectly genomic DNA!!! The cDNA is ok because I am able to amplify other transcripts. Since the fragment has 1kb we also tried RNAseH treatment but this did not help. I tried even to order a new stock of the same primers, but it didn't change anything. Finally we also tried to amplify old cDNA samples in which we managed to amplify successfully this transcrip before and now IT DOES NOT work!!!!!! I'm REALLY puzzled, does anybody has any hint on this problem?


Thanks a lot,
ClŠudia



#3 vitalgene

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Posted 04 August 2011 - 05:01 AM

i look back with a similar experience, however it was solved by a crazy modification. The regular PCRs I used 20ul reaction volume, but in the modifiedPCR I used 100ul reaction and the bands were indeed perfekt, Sounds funny huh

#4 csmartinho

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Posted 04 August 2011 - 07:55 AM

waw, I would never think about that, I don't know the reason for that! I will definitely try it out since at the moment I'm lacking new ideas. If it works that's great. THANKS A LOT for the suggestion :)




i look back with a similar experience, however it was solved by a crazy modification. The regular PCRs I used 20ul reaction volume, but in the modifiedPCR I used 100ul reaction and the bands were indeed perfekt, Sounds funny huh






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