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Weird PCR results


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#1 Nephrite

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Posted 03 April 2018 - 01:35 AM

Hello!

I try to characterize expression of some enzymes in skeletal muscle in control and experimental samples from mice.

I started this research as a small experiment some years ago in another laboratory, I got convincing results, which were checked by more competent colleagues, and now I expand this study in my laboratory.

Most of the chemicals and the primer sequences are the same, mice are ICR like before.

Before going further I decided to reproduce the previous experiment.

I was shocked to see amplification product in my control samples, where there shouldn`t be anything. Absolutely anything.

I was hypershocked to see no or very low amplification in my experimental samples, which is opposite to my previous results.

Could anybody possibly have an idea why is this happening?



#2 bob1

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Posted 03 April 2018 - 06:23 AM

There are a few things you can try - double check the conditions you used in your old lab - even such a minor thing as a difference in PCR machine can sometimes cause this type of difference. You can also check the strain of mouse (possible to genotype fully?). You might want to consider having external controls for the PCR - maybe plasmids with your primer binding sites but non-specific sequence of a different size to that of your current PCR between the binding sites - so that you can test the conditions and know that there are no contamination issues.

 

As to failing/weak PCR - there are a ton of things that could be going wrong. The usual causes are: starting material (try adding more and diluting it out), annealing temperature, and magnesium concentration.



#3 Nephrite

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Posted 04 April 2018 - 03:59 AM

Thank you, Bob1!






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