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PCR inconsistency


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#1 lightbird

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Posted 26 January 2010 - 02:26 AM

Hi, I'm new to the forums and have a prolonged PCR problem.

I'm working on a statistic about the TP53 gene Pro72Arg polymorphism frequency in melanoma and control patients in my country for my Bachelor thesis and need to amplify a ~250 bp fragment of the 4th exon. However, no matter how carefully I check the volumes and the pressure applied to the micropippete (the first-second stop bit), I often tend to get excess reaction mix left in the tube after filling in the PCR tubes. Moreover, the PCR itself is pretty inconsistent, sometimes I get the product, sometimes I don't, and it's wearing out both me and my tutor, because I just can't obtain enough material to work with for the statistic.

We use a cycle of 94C for 30s (melting), 62C for 30s (primer annealing), 72C for 1 minute (synthesis), and this protocol works when my tutor or my elder student colleague is making the reaction, but not with me. I've also checked that it's not a problem of a faulty electrophoresis. What could I possibly do to ensure a positive outcome?

#2 The Question

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Posted 26 January 2010 - 04:30 AM

Hi, I'm new to the forums and have a prolonged PCR problem.

I'm working on a statistic about the TP53 gene Pro72Arg polymorphism frequency in melanoma and control patients in my country for my Bachelor thesis and need to amplify a ~250 bp fragment of the 4th exon. However, no matter how carefully I check the volumes and the pressure applied to the micropippete (the first-second stop bit), I often tend to get excess reaction mix left in the tube after filling in the PCR tubes. Moreover, the PCR itself is pretty inconsistent, sometimes I get the product, sometimes I don't, and it's wearing out both me and my tutor, because I just can't obtain enough material to work with for the statistic.

We use a cycle of 94C for 30s (melting), 62C for 30s (primer annealing), 72C for 1 minute (synthesis), and this protocol works when my tutor or my elder student colleague is making the reaction, but not with me. I've also checked that it's not a problem of a faulty electrophoresis. What could I possibly do to ensure a positive outcome?




Hi Lightbird

Inconsistency in PCR reaction can occur due to several factors. Proper use of micropipettes is important but after several attempts is should be ok, I suppose. Are you using the same reagents which your tutor and elder student colleagues are using? Problem in your reagents can cause inconsistency in your PCR reaction. Try the ones your tutor is using (if you are using others at this moment).

Good Luck
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#3 nightingale

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Posted 26 January 2010 - 12:52 PM

hello,
my advice is to watch your tutor or your elder student colleague carefully when they are to run the samples...
tiny details do make difference, as vortexing / good mixing the samples before
taking the required volume..

in ur notebook write down everything they do ..
and then give it a try..

good luck and Best Wishes

Edited by nightingale, 26 January 2010 - 12:53 PM.

" The more you learn, the more you realize how little you know ... "

#4 Mighty Mouse

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Posted 26 January 2010 - 08:18 PM

I often tend to get excess reaction mix left in the tube after filling in the PCR tubes.


This is fine, typically when you make your master mix for a PCR you make a little more than is necessary as a small amount above what you pipette out is lost each time you pipette (this is unavoidable). So having a little extra is expected.

I agree with the previous comments. Some other keys are to keep everything on ice, and make sure the master mix is well mixed (either via pipetting or vortexing then centrifuging) prior to pipetting into the PCR tubes.

MM
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#5 lightbird

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Posted 28 January 2010 - 05:57 AM

Thanks for the comments. The problem is, I've watched my tutor and elder colleague many times and I always take note on what I do, whether anything is added etc., but I still can't get a result. Also, they have tried to do the run with the same reagents and have succeeded, so I still have no clue what might have been done wrong..




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