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Asking for help with electrophoresis

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Asking for help with electrophoresis

Postby ll2013 » Thu Jun 06, 2013 1:09 am

Dear all, my lab has some problems with DNA agarose gel electrophoresis.

First, the small DNAs cannot be separated. We are using BioLine hyperladder II as marker, which has bands from 50bp to 2000bp. The separation from 300bp to 2000bp is just fine. However, the 50bp, 100bp, 200bp and 300bp bands have weird running pattern. They were quite close to each other, look like a pressed spring when comparing with the bigger bands.

Second, the samples on the side of the gel look like being “drifted down”. If I ran same samples for all wells, they would show a “sad face” pattern.

The gels were ran with: 1.2% ~1.4% agarose gel, 0.5X TBE buffer (I made the buffer twice, similar results), 4~5 V/cm, and the room temperature was absolutely cool. The gels were not extremely warm during electrophoresis.

May anyone help me to analyze the problem? A gel photo is attached with the sample DNA marker image to show the difference. Thanks a lot!
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Postby JackBean » Thu Jun 06, 2013 9:53 pm

I don't think you will be able to resolve 50-300 bp with 1.4% agarose. Since you have the same band in all samples. I'd say your dye goes just in that region, thus you do not see the respective bands.
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Postby Cat » Sat Jun 08, 2013 3:57 pm

1. It seems that you are using regular agarose which separates 200bp and up. You need genetic analysis grade agarose that allows good separation of fragments bellow 1kb. And even with that, you will need to make a 2-3% agarose gel. Here is the link to the agarose that you need: http://www.fishersci.com/ecomm/servlet/ ... hType=PROD

2. Uneven running pattern on the gel = incompletely melted agarose or gel had clamps when it was poored. Make sure that gel that you poor is clear and contains no particles.

Good luck!
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Postby JackBean » Sat Jun 08, 2013 4:45 pm

ll2013 doesn't need any special agarose, just higher percentage, if wants to see bands 50-300 bp. But than will have problems with the bigger bands.
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Postby Cat » Sun Jun 09, 2013 4:47 pm

Good resolution of the bands over 1kb on the gel in the picture tells me that regular agarose was used - resolving power of that is usually 200bp and up. Increasing the percentage of agarose would improve resolution of smaller fragments somewhat, but if GOOD resolution is required, genetic grade agarose is needed.
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Postby ll2013 » Mon Jun 17, 2013 1:51 pm

Thanks for all of your help!
I did more gel electrophoresis and got quite good results this time. Below was what I changed:
1. 1.8% agarose gel, but still regular agarose
2. When melting the agarose, instead of using microwave, this time I used a heating block with stirring.
3. Used a much bigger electrophoresis tank with buffer circulation. The voltage was decreased to 2~3V/cm.

It is hard to say which was the critical change, but the overall result was good. My main targets are 200bp~400bp, so the current resolution is good enough for me --- of course the running time is much longer.

Again, thanks for your help! Good luck with all your experiments!
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