PCR stopped working

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Syma's picture
PCR stopped working

I had been successfully running PCR,  able to clone PCR products, identify alternatively spliced transcripts and get them sequenced. I have gotten into trouble since last september.  Have successfully isolated and identified POLB and TRPV1  transcript variants from total breast cancer RNA.  Then i started working on isolation and identification if INSR splice variants. Although literature says a lot about INSR's increased expression in carcinomas, liver, fat, and muscles... i was only able to amplify, isolate and identify few transcripts of last part of gene, from total liver RNA.  Things stopped working all at once. we trouble-shooted and trouble-shooted. Even the genes that had been amplified and had shown bands on gel before were not showing up. Only the positives show up always. 

  • Have checked the validity and concentration of our RNAs, by nanospec. They all are good quality commercial RNAs.
  • Have checked the validity of cDNAs by real time PCR. The results showed the presence of genes in the sample cDNAs.
  • Have tried different PCRprotocols & PCR  programs (ones that had given us results in the past, the manufacturer's protocol and recommended prog, gradient, touchdown etc)
  • Have changed Taq from the one that we used to use and have always gotten good results ( thermostable Taq DNA Polymerase) to Platinum Taq
  • Change of Taq only helped in getting POLB from brain, breast cancer and liver RNAs
  • But still stuck at getting TRPV1 and INSR from them... that had shown up in them almost a year ago.
  • Can anybody please help me out & advise me about what further steps to take. What am I missing? Wher am I making mistake(s)? Now it has drained my nerves, and I have only 2 months to show some results to my committee