inconsistent PCR - (Aug/08/2005 )
Hello everyone. I've been getting some inconsistent PCR results lately. I am performing methylated specific pcr using a touchdown protocol in a GeneAmp 9700. The problem is that my PCR as of late has become a roll the dice event in that sometimes it works and others it doesn't given the same conditions and concentrations of reagents. I've tried playing with my primers switching dNTP and DNA concentrations. Another phenomenon
I've noticed with my reaction is that it works better if I don't mix the reaction after adding my DNA to the mastermix. If anyone has any comments, suggestions I would really appreciate your help. Thank you very much
I've noticed with my reaction is that it works better if I don't mix the reaction after adding my DNA to the mastermix. If anyone has any comments, suggestions I would really appreciate your help. Thank you very much
Hi there, I am having the same problem. I use HotStar Mastermix for all PCR's. My PCR was working just fine a few months back but one day it just stopped working.I have no Idea what to think.
Hope somebody can help us.We are in the same boat.
Have you tried using a different thermal cycler?
Could it be the prob with PCR reagents? I had the same prob last time, then I found out that how I prepare the DNA template also will affect the PCR result. And sometimes it's just the reagents are expired. I dont think mixing is the source of error, I always mix mine and spin all the solution down before putting into the thermo cycler. How about ur controls? Positive and negative controls?
However...I did 3 PCR at the same time, with exactly the same reagents and thermocycle, but still receiving inconsistent results!!
Maybe your reagents or the water are being contaminated haha
That's the only reason I can think of 
YS I was curious as to how your prep affected your pcr could you explain further? I also tried different thermocyclers and its wierd because some times even with the same reagents and all I get various intensities of my bands.
I think various intensities of the band is still much better than getting bands at the wrong size or even not getting a band while u should.
For me, when I get a bacteria strain that gives me the desired band size in PCR, I will keep that in mind that it could be used as positive control. However, sometimes these so-called positive controls show diff band size and worse, the band disappear. In this case, I usually just change the sterile water and I used in PCR preparation, or try a fresh tube of buffer and MgCl2.