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when does pcr products degrade like this? - images attached (Jul/08/2008 )

Can anybody suggest me when does this kind of thing (I assume degradation) happens in PCR?
I'm trying to clone out genes from cDNA. When I did this first time, it worked; but now its not working, whatever I do.
I tried decreasing the amount of starting cDNA, decreasing the number of PCR cycles, using fresh cDNA every time, decreasing the annealing temperature much much lower than the theoretical annealing temperature, increasing the polymerization time, decreasing the primer concentration, increasing the reaction quantity, and all I get is a smear like this. The working conditions are 2ul cDNA (prepared from 2ug RNA in a 25ul reaction volume), 100-150ng primers, promega go-taq green master mix, and the PCR conditions are as follows:
94C for 10 mins
94 for 20 secs
58 for 30 secs (the lowest Tm of my primers is 61C)
72 for 2 mins (the length of the largest gene is 1.1kb)
35 cycles
72 for 10 mins
4 forever.

-jangajarn-

It is probably not degradation, as I doubt there are any DNases that would work at those temperatures. Can you explain what the lanes in the gel are, and where the expected band should be?
dan

-rosewater-

thanks for the help. the lanes are different genes (lane 1 &2 correspond to the same gene, lane 4&5 are the same, and so on). The expected size are gene#1-604 bp, #2-517, #3-829bp, and #4-751bp. the ladder is a 100bp ladder(from lowest to highest: 100, 250, 500, 750, 1000 and 2000bp).

-jangajarn-

I think you are loading too much DNA onto your gel. Try the gel with 10x less of your PCR product. That's a 1 Kb max size ladder, right? You may want to increase, not decrease the annealing temperature. You could also try decreasing the amount of cDNA you add, which can sometimes be a problem.

-phage434-

I would do two things:

PCR gradient and MgCl2 concentration optimization... then you could also reduced the initial denaturation step, 10 minutes is too long for cDNA. As I understood, you are using the same PCR program for different product length ? don't do this, and 2 minutes extension is too long for nowadays pol, use 1 minute for 1.1Kb. of course if you amplify a 1.1Kb fragment during 2 minutes and only this fragment appears, it means that your PCR is very specific, so it's okay, but it's obviously not your case.

ps: could you produce a non-RT'ed PCR on your RNA extraction ?

-ph3no-

I agree with the above with regards to testing different temps and Mg. Also do some controls with one primer omitted from the reaction. Maybe some of the crap is due to one prime hybridizing at an unexpected site.
Also check out http://www.molbiol.ru/eng/scripts/01_14.html

-rosewater-

Thanks for the comments. I'm using a ready-to-use PCR mix (promega go-taq green master mix). And sorry, the initial denaturation was not 10 minutes. Its 4 minutes.
What I find strange is that these conditions worked perfectly initially. But now, I dont know what happened, its not working. Would using 500ng poly DT for RT (with 2ug RNA) be the reason for it?
Thanks again..

-jangajarn-

QUOTE (jangajarn @ Jul 10 2008, 11:52 PM)
Thanks for the comments. I'm using a ready-to-use PCR mix (promega go-taq green master mix). And sorry, the initial denaturation was not 10 minutes. Its 4 minutes.
What I find strange is that these conditions worked perfectly initially. But now, I dont know what happened, its not working. Would using 500ng poly DT for RT (with 2ug RNA) be the reason for it?
Thanks again..


500ng of poly-dT ? I don+t think so, the fermentas kit uses such amounts, it might be that using anchored dT could improve specificity...

-ph3no-