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DNA size band increased after gel electrophoresis with Quaigen fit

microbiology PCR purification

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4 replies to this topic

#1 myandas

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Posted 27 February 2013 - 06:37 AM

Hi dear.
I'm doing DNA extraction and purification for pyrosequencing. I did gel electrophoresis extraction by low-temp-melting agarose gel and cut the DNA and put into tube then used Quaigen kit for purification. Then I checked DNA recovery rate, it was around 35%. Then I checked DNA size and any contamination by Bioanalyzer 2100, but it showed very interesting image.
Before purification, my sample was around 600 bp, but after purification it increased over 700 bp. Initially I thought I made mistake or did something wrong. Then I did purification twice. But all results are same as previous one. Before purification everything is ok, but after purification, DNA size increased. I don't understand what is going on.
Can anyone tell me why and is it Ok or not?Screen Shot 2013-02-27 at 11.31.45 PM.png

#2 jerryshelly1

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Posted 27 February 2013 - 11:21 AM

If I were to guess, it looks like your sample is more concentrated which could lead to a small shift. Read the concentration on each loaded sample and load equimolar ratios.

Good luck

#3 bob1

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Posted 27 February 2013 - 11:21 AM

The image you show shows bands of less than 700 bp... On an ordinary gel you could easily interpret these as being about 600 bp depending on how well you have resolved and separated the fragments of your ladder.

#4 phage434

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Posted 27 February 2013 - 12:49 PM

The salt concentration of your buffer also affects the band position. You may have purified the DNA from a buffer containing salt, into a low salt buffer.

#5 myandas

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Posted 27 February 2013 - 09:10 PM

Thank you very much for all. I will consider all the answers.





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