For my experiments, I have to express ectodomains of human CD28 and CD3z. As a source, I extracted RNA from Jurkat cells and verified the quality of RNA on the gel. I, then, prepared cDNA using oligodTs. However, when I tried to amplify from the cDNA using gene-specific primers of CD28 and CD3z ectodomains, I did not get any amplification. Of the 25uL cDNA reaction, I tried amplification with 0.1, 0.2, 0.4, 0.6, 1.0, 2.0, and 4.0 uL as template in 10/20uL PCR mix. I was able to get amplification using Beta-actin primers with 0.1, 0.2 and 0.4uL of cDNA as template.
I again prepared cDNA using gene specific primers and used 2uL of 25uL cDNA reaction as a template in a 20uL PCR mix. Again, I did not get any amplification.
What could be the possible way to go around this problem ?
Have you confirmed the primers work (in other cells or positive plasmid)?
Yes , you have a problem with yours primers. Did you test them at different temperatures ? Did you test them in genomic ? If it is possible
Thank You Junjie Shao and Guy Longepied for your suggestions. I do not have a positive control such as the same genes cloned in a plasmid. I have just revived Jurkat cells from the stock and waiting for them to grow good enough. As suggested by Guy Longepied , I am planning to isolate the genomic DNA alongside the Trizol based purification of RNA. I am then planning to setup temperature gradients at different genomic DNA concentrations for both pairs of primers. I hope the genomic DNA can act as a positive control. Will let you know when I set up these PCRs.
Thank You again for your suggestions.
For isolation DNA from Jurkat Cell don't use Trizol. The best way is to use proteinase K . Go on PubMed to find the article : Simplified mammalian DNA isolation procedure. Peter W. Laird . Nucleic Acids Research vol.19, N° 15 1991
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