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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Reverse PCR
Posted by: frederic.marrache (IP Hidden, New member, 2)
Date: July 17, 2006 12:47PM
1 / I am trying to detect a transgene in a transgenic mouse by Reverse Transcritpion PCR. The mouse has a clear phenotype that makes me believe she ex[ress the transgene. Although my PCR is working on genomic and plasmidic DNA, there is no way I get it to wprk on my cDNA (that appear to be of good quality). GAPDH PCR is working. I initailly had a smear, that disappear witt DMSO, but still no band. I also changed temperature and Mg concentration. Any idea?
2 / I am trying to do reverse transcitpion for an other project on mammary tissue. The only thing i manage to get is a smear, for GAPDH. The reaction is working for all other oragsn tested (gut pancreas spleen liver...).
Re: Reverse PCR
Posted by: TerAle (IP Hidden, New member, 5)
Date: July 19, 2006 01:23PM
1. Once using a new pack of DNA polymerase helped me in the same situation.
2. DNA contamination (sometimes is a problem in particular tissues). 3. I'm sorry for that suggestion, but, if it works on a genomic DNA, could one of your primers by any chance correspond to an intronic region which would not show up in cDNA? Al.
Re: Reverse PCR
Posted by: frederic.marrache (IP Hidden, New member, 2)
Date: July 19, 2006 02:41PM
Thank you
1/ Why not 2/ Unlikely since I do DNAses treatment and that the control without reverse transriptase is absolutely negative (wheras I have a smear in my sample) 3/ Unlikely as it is a transgene
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