My PCR just stopped working....

adagnall

New member
Nov 16, 2015
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#1
The title says it all really. Im doing some Bisulphite treatment of my gene of interest. The PCR worked fine, ran it on a gel, excised, cleaned, cloned into pGEM, transformed, picked colonies, sent for sequencing all worked, no problem. Repeated on new DNA sample, no bands. Tried a positve control using a clone which I had sequenced and could see had my primer sites in it. No bands. Realiquotted primers. No Bands. Taq works fine for other PCRs no problem. But changed, MG, Taq and cycling conditions. No bands. Re-ordered primers. No bands. Tried denaturing primers and snap cooling them, if secondary structure was a problem. No bands. Used same lanes in PCR machine for other pcrs which worked so not a machin problem. Im pretty stumped.
 

macbride

New member
Nov 16, 2015
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#3
Are you using different aliquots of sterile water or PCR buffer from other people in your lab (for whom PCR continues to work)? I'm just trying to eliminate all the possibilities...maybe you got contamination somewhere in your supplies. I like to add a few % DMSO to PCRs when they are being tricky. We also keep a few different polymerases around, so I try others when I'm having problems.
 

nin1318

New member
Nov 16, 2015
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#4
i would try a new aloquot of everything...mgcl2, dntp, pcr buffer, water...you may not know exactly what the problem was, but i f you know the taq is good that is the only expensive part so just use new bits of the others.
 

frasermoss

Administrator
Staff member
Nov 16, 2015
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#5
Oh the joys of Mol Biol! I feel your pain!

Maybe in this instance you need MSP primers instead of conventional bispulphite primers, or visa versa depending on what you are using now.

Also you might want design some nested primers to increase you sepcificity and/yield.

Maybe you should double check all your paramters yet again, and use a guide such as

Clark SJ, Harrison J, Paul CL, Frommer M.
High sensitivity mapping of methylated cytosines.
Nucleic Acids Res. 1994 Aug 11;22(15):2990-7.

to help you with troubleshooting.

good luck
 

frasermoss

Administrator
Staff member
Nov 16, 2015
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#6
this thread at protocols-online.org may also help

http://www.protocol-online.org/forums/lofiversion/index.php/f20.html
 

camembert

New member
Nov 16, 2015
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#7
I already had the same problem, and after some "research" I found that My DNA was from a bad quality, so I extracted it again using a different kit. Never had THIS problem again, but lots of others. So succes.

I'm using a DNA extraction kit of Qiagen. A bit expensive but verry good kits.
 

rtbenn

New member
Nov 16, 2015
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#8
try re-ordering your primers. long story but sometimes primers unexplicably degradate quickly... good luck, looks like you have a bit of trouble shooting ahead of you.
 
Oct 27, 2016
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#9

Hello there,

I realise this is a very old post, but did you ever get to the bottom of this issue? I'm having the exact same problem myself, but with 80 assays which previously worked and are now giving inconsistent results!