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PCR woes


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6 replies to this topic

#1 pcrpetie

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Posted 01 March 2005 - 03:57 PM

Help! I run several pcr assays in a clinical/developmental lab. About two weeks ago I began to have a problem with intermittent pcr failure. The problem was localized to one or a few rxn tubes in a run. The same master mix goes into all of the rxn tubes plus the template DNA. The particularly quirky part is that I run two lanes for each sample (same template); the rxn might work in one tube but not the next.
I know what you're thinking ... this guy can't pipette, but I'm positive that I am getting template into every rxn tube. I even mix the template each time I add it to a tube.
I have replaced all of the reagents in the pcr reaction. MgCl2, 10 x buffer, and taq polm.
I do not believe that the problem is with my extractions; I have one assay that seems to be unaffected.
Any suggestions?

#2 sharath

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Posted 01 March 2005 - 08:46 PM

Are you sure you are loading equal volume of the reaction mix in both the wells. I had noticed this behaviour while running a SDS PAGE gel. I had loaded protein sample from the same tube in 2 wells. But, while staining with silver nitrate, only one of them gave a nice band and the other failed. I figured out that while loading there was an irregularity. The failed one contained slightly less sample than the other. As the concetnration was at the critical for staining, the lowered one fialed to appear.

Hope this explains your problem too.

Edited by sharath, 01 March 2005 - 08:47 PM.

Sharath B.

#3 jadefalcon

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Posted 02 March 2005 - 04:00 AM

one (though not very likely - I heard about this only once) possibility to think of is the cycler you use... we had to check our's some time ago, and found that two specific places for tubes had alway slightly less temperature that the others. so when using all places in the cylcer, transcription efficiency was worse in the tubes placed in those two places compared to the others.

this possibility can be checked easily and quickly by running some test pcrs, containing all the same mastermix + template, but are run in differnt places of your cycler. If you see no difference in amplification afterwards, this possibilty can be ruled out.

mike
--- He who finds typos may keep them! ---

#4 pcrpetie

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Posted 02 March 2005 - 05:48 AM

Sharath, I'd like to think that my technique is not the problem... however as my p200 does not always form a good seal with the pipett tip I cannot rule out your suggestion. I've been paying a great deal of attention to the template volume, but I can't say the same for the rxn mix.

Mike, I've had the same concern and will do an experiment, though I do have two thermocyclers and the problem has occured with both.

What about letting my template DNA get to dry after the 70% ETOH rinse before adding hydration buffer? This thought occured to me last night.

#5 fred_33

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Posted 02 March 2005 - 06:06 AM

hi
according to the fact all your samples should get the same conditions / time of drying, this is probably not the source of the problem... sorry but maybe you can let them dry whithout heating them (it changes result from my sequencing experiments regarding efficiency)...
if you think your P200 may be a problem, wash it good (sonication if a grat procedure for the plastic part of the pipette) and see if changes occurs.

fred

#6 lula

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Posted 04 March 2005 - 09:19 AM

hello
in the past period ur lab has also had several PCR failures and guess what was theproblem,,,,thethermocycler weare using had little oil left in the test tube in the block
this caused differences between the block heat and the tube of the product temperature ....just adding little oil gave us good results

#7 pcrpetie

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Posted 09 March 2005 - 05:58 AM

Sharath, Mike, Lula, Fred,
Thanks for the advice. I swithched from Taq to Amplitaq and my woes went away.
Jim




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