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problem with Rt-PCR


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#1 helene

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Posted 01 February 2002 - 12:23 PM

I'm trying to separate four mRNA by RT-PCR.These mRNA are the result of an alternative splicing, so they have aproximatively the same lenght. Furthemore, the two introns are localized at the edge of the mRNA.
I've tryed several different taq,primers, t°annealing, Magnesium concentration without any result. Please help me!
              Helene
PS: sorry for my very bad english!

#2 Cthulhu

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Posted 01 February 2002 - 01:31 PM

If I understand you correctly you want to amplify one of the four cDNA sequences, without amplifying the other three at the same time. This problem can be solved through careful PCR primer design, where you make primers for sequences where the four differ. You will need five different primers. For instance, the 5' primer can be the same for all four cDNAs, while the 3' primers are different. The 3' primers, in this case, should be based on exons that are unique in each of the differentially spliced mRNAs. If you choose to amplify all four at the same time you could maybe separate them with the right concentration of agarose in the gel, based on that they differ in length, or if you could design probes that are unique to each of the four cDNAs, you could tell them apart by Southern hybridization to the PCR product.




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