PCR & Agarose gel electrophoresis

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Juanita1
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PCR & Agarose gel electrophoresis

Post by Juanita1 » Thu Aug 20, 2009 8:21 pm

Hi there,

I have been trying to determine the genotype of some cell samples using PCR and agaorse gel electrophoresis. On one of the samples, I was supposed to find two bands representing a heterozygote for a particular mutated receptor but I could only see one band representing the wild type homozygote.

When other people did this experiment, they correclt yobserved 2 bands.

Now I need to do a write up of this and why my particular experiment went wrong or what could have been the reasons.

Can anyone help me as to what kind of things can go wrong with PCR and agarose gel electrophoresis experiments. The only thing I can think of is contamination of DNA samples but there must be other reasons. I would be grateful if someone can help.

Thanks

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MrMistery
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Post by MrMistery » Thu Aug 20, 2009 9:35 pm

how big were the differnces in size? If they are very big maybe you just ran the gel too fast or too long and the smaller band ran off the gel.
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apolll
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Re: PCR & Agarose gel electrophoresis

Post by apolll » Sun Aug 23, 2009 8:40 pm

Hi!
Could you describe the experiment more detailed? where do the primers anneal? why are there two bands? did you use more than 2 primers? which polymerase did you use (proofreading or not)? does a primer anneal to the mutated site?
I don't think that contamination is the explanation to that. I'd expect more bands not too less if you had a contamination. In my opinion the only thinkable explanation involving a contamination would be if the extraction of the cellular DNA went wrong. Do you have the gene already in a plasmid anywhere in your lab? We once found out that we had plasmid contamination in autoclaved water. When you use this water for PCR, you get a band... If the water was contaminated with such a plasmid and your DNA extraction didn't work properly, this could be an explanation. Did you do a beta-actin or other houskeeping-gene control? If this worked your extracted DNA should be fine.
How often did you repeat the experiment? Can you outrule that you just messed up? (Sorry, but this happens to me sometimes :wink: )
Did you use the same DNA extraction as you colleagues? the same PCR machine? the exact same PCR programme? Did you see some DNA stuck in the pockets of the gel?
Sorry, so far I have more questions than answers...

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jyaron
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Post by jyaron » Sat Sep 05, 2009 10:32 pm

Where your thermal cycle times the same as theirs? What about the voltage across the gel and the time of the run?

Did you expose the RNA to light too much? It's possible you denatured them. Also, there may have been RNAses in your extract that could have reduced copy count drastically.
Experience: Cell Biology, Confocal Microscopy, Developmental Biology, Evolutionary Biology, Genetics, Genomics, Physiology

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