problem in PCR pdt size - (Jul/08/2008 )
Hi,
I have designed a few intron spanning primers to standardize RT-PCR. Data from ensembl gives a higher amplicon size from gDNA and smaller from cDNA. On PCR from gDNA, the pdt sizes are wierd. sometimes im getting pdt size of cDNA generated amplicon and sometimes it is of higher size than the gDNA generated amplicon size. but pcr with cDNA gives the correct pdt size. Ive done a gradient pcr, used hot start and taq pol & tried various other combinations. Can ne body help me out with this...
Are you sure you've removed gDNA from your RT template RNA? Could be why you're getting gDNA sized band in you cDNA.
Similarly, are you sure you don't have RNA in you're gDNA prep?
Finally is it possible that you might have carried one template into the other (or stock reagent tubes) while setting up your reactions? Try running a PCR w/o either template. cheers-JAH
Similarly, are you sure you don't have RNA in you're gDNA prep?
Finally is it possible that you might have carried one template into the other (or stock reagent tubes) while setting up your reactions? Try running a PCR w/o either template. cheers-JAH
Hi Jah,
Thanks for ur reply. I gave a DNase treatment to RNA before setting RT. I'm having no problem wit PCR from cdna. only problem is when i use the same set of primers wit gdna. But as u have suggested, ill give an RNase treatment in gdna and see wht results i get. Also im setting pcr with individual templates, so as 2 not confuse wit cdna & gdna. My worry is that the major product in pcr wit gdna is that im getting a pdt size matching to cdna. Ill try out RNase treatment. Thankyou.
What type of sample do the gDNA come from??
My gDNA template is from NIH (mouse) and HeLa (human) cells