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Gel Electrophoresis....


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5 replies to this topic

#1 gebirgsziege

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Posted 27 January 2009 - 06:54 AM

Maybe somebody can help me out of my confusion: I just loaded a 1% Agarose Gel (TAE) with 32 samples at 5V/cm. When I came back to check if everything is ok, I could not belive my eyes: 4 out of the 32 samples were running in the wrong direction!
All the other samples were running from - to +. All samples were PCRed with the same chemicals at the same time by the same person :(

Anybody an idea what could have happened there?????? And why only 4 samples were going the wrong way????
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#2 mdfenko

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Posted 27 January 2009 - 08:15 AM

very strange, i've never seen it happen with nucleic acid electrophoresis.

is there anything that was different about how those samples were prepared (eg pH) that might have caused a charge reversal of the nucleic acid? or neutralization?

if you somehow neutralized the nucleic acid (eg by creating a salt) then the migration may have been by electroosmotic flow instead of by charge. eof generally runs from + to -.

if you used a low eof agarose then the sample may have remained at the origin.
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#3 gebirgsziege

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Posted 27 January 2009 - 08:56 AM

I do not think that something changed the pH, but I think it is the only rational explaination....although all samples were treaten the same (same extraction, cleanup, PCR, loading dye....)

But the four samples really were running from + to - including both dyes from the loading buffer......and I tried again: same picture :(
very strange!


But thanks for your suggestion, it sounds rational. Still puzzeld how something can happen!
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#4 perneseblue

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Posted 27 January 2009 - 09:49 AM

very strange. Are you running a vertical gel? Could it be a problem of ethanol contamination?
May your PCR products be long, your protocols short and your boss on holiday

#5 pcrboy

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Posted 27 January 2009 - 11:31 AM

Yes, it's quite strange. When you repeated were they the same wells? If its the one on the edges, maybe something is disfunctional with your gel chamber, such that you wont get an even distribution of current.

#6 gebirgsziege

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Posted 27 January 2009 - 11:23 PM

There seems to have somthing happened with this samples: not same wells, I was thinking of a broken divice :(

no its a horizontal gel......and I do not think that its EtOH contamination, but I repeated the PCR and hope it works this time....and will repeat the extraction, maybe just some sort of my samples feeling the snow coming :angry:
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)




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