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Smears in Bisulfite seq. PCR - (Jul/09/2010 )

Hi everybody,
I have a smear problem in my Bisulfite PCR. I have done this PCR with same primers, it was giving a band but it was also giving some smear in gel. But now only thing i can get is smear, i have changed buffer, Mg, primers, PCR water but i still have smears even in negative control <_< I have tried doing with a different master mix from another company to see if my samples were problematic, as a result i got no smear but no band neither. I really need some help. Thanx.

-epibet-

Smear is very common in bisulfite PCR. Try the following remeadies to see if they help.

1. Hotstart your PCR. Assemble your PCR reaction and immediately transfer your tubes to your PCR machine preheated at 94C. You can also use hotstart taq. I found JumpStart RedTaq from Sigma helps a lot.

2. Try several different annealing temperatures aroud 55C.

3. If the above does not solve the problem, redesign your PCR primers.

4. Search smear + bisulfite PCR in this forum.

-pcrman-

pcrman on Jul 11 2010, 08:15 AM said:

Smear is very common in bisulfite PCR. Try the following remeadies to see if they help.

1. Hotstart your PCR. Assemble your PCR reaction and immediately transfer your tubes to your PCR machine preheated at 94C. You can also use hotstart taq. I found JumpStart RedTaq from Sigma helps a lot.

2. Try several different annealing temperatures aroud 55C.

3. If the above does not solve the problem, redesign your PCR primers.

4. Search smear + bisulfite PCR in this forum.


Thanx pcrman i got my faint bants back :) i preheated the pcr machine to 94C

-epibet-

epibet on Fri Jul 9 13:02:14 2010 said:


Hi everybody,
I have a smear problem in my Bisulfite PCR. I have done this PCR with same primers, it was giving a band but it was also giving some smear in gel. But now only thing i can get is smear, i have changed buffer, Mg, primers, PCR water but i still have smears even in negative control <_< I have tried doing with a different master mix from another company to see if my samples were problematic, as a result i got no smear but no band neither. I really need some help. Thanx.

Hi

Im new in the forum and ive read your problem and i have almost the same problem but even ive seen a band in the negative control the samples are so clean without smear but the negative is the problem ive changed all reactives even with new primers but a i have the same problem and im really desperate so my question is: you solved your problem? and if you did, how was it? and please tel me if you have any idea of what is happening in my caseim really desperate.

Thanks a lot

gatto

-gatto-