pyrosequencing issue - (Jun/12/2007 )
I have a problem with two pyro assays... one time when I run my pcr I get bands for some of my samples, then another time, I just get smears for the same samples. Why would this be happening? 
Hi Bunny,
can you give us some more details on your PCR?
Bram
can you give us some more details on your PCR?
Bram
Sorry, this is just so frustrating. I am running PCR with bisulfite converted samples and my F primer is biotinylated. I store my bis.conv. DNA at -20 and -80. PCR is standard/normal pcr, annealing temp. determined after running a gradient.
I am running it one day and it works out great... then the next day...same conditions, same stocks, same samples, same anything, it does not work anymore. This means some samples do not give any signal anymore, which gave nice signal the day before under the same conditions. (EEEEEEK!!) The I run again a few times, during the next few day and PCR gets progressivly worse, where I only get smears at a point. Then I wait a few weeks, run the same thing again... and poof some samples now give nice signal again... but not all of them. I have no explanation for this, as other people in my lab use the same assay without issues. My samples work fine for other assays (other genes)...
Plus if my samples were degarded, why does it work again after a few weeks, and for other genes?
thanx!
Hi e-bunny,
We have had very similar problems to yourself, in that the PCR of our bisulphite treated DNA (used EZ kit) were hit and miss in regards to obtaining a product. We even tried a couple of different PCR blocks and this didn't work either. However, we recently tried a PCR block in another lab and have much more success with the PCR. We think that maybe this PCR machine is more accurate and hence the reactions are working better. I can't think of any other explanation. It could be that all wells in the heating block aren't useable? I know this is a shot in the dark but it might be worth trying a few other machines, especially ones which have been serviced regularly.
If we still have a problem with certain samples, it usually helps to vary the amount of template, probably use less to improve the smeared samples. Hope this is useful
Orinocho
We have had very similar problems to yourself, in that the PCR of our bisulphite treated DNA (used EZ kit) were hit and miss in regards to obtaining a product. We even tried a couple of different PCR blocks and this didn't work either. However, we recently tried a PCR block in another lab and have much more success with the PCR. We think that maybe this PCR machine is more accurate and hence the reactions are working better. I can't think of any other explanation. It could be that all wells in the heating block aren't useable? I know this is a shot in the dark but it might be worth trying a few other machines, especially ones which have been serviced regularly.
If we still have a problem with certain samples, it usually helps to vary the amount of template, probably use less to improve the smeared samples. Hope this is useful
Orinocho
Hi, not such a dark shot. I have just been having similar problems with standard PCR. Worked one day, not the other. same sample conditions etc... then, prepared reaction in duplicate and run it in separate PCR machines, guess, my machine did not worked. It was then suggested to me to move the PCR to a different corner in the lab (hence different power supply), I did laugh at this, but... guess again. IT HAS SOLVED MY PROBLEMS, and now PCR works every time.
I know it sounds weird, I'm still stunned, but I promise is true.