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need help with my gel

need help with my gel - PCR - Polymerase Chain Reaction Forum

need help with my gel - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 02-07-2012, 12:33 AM
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Default need help with my gel



I need help with my gel. I made a PCR product of 20ul total, ran 3ul of the product on a 2% agarose pre-cast gel. This is the first picture. There is a nice band at the correct bp size (400bp), but a fainter band is above it. So I wanted to gel purify out my correct band.

I ran the 17ul leftover on a poured 2.5% TBE gel, at 100V for 40minutes. The DNA ladder on the right lane is a 100bp ladder, so my product is 4 bands from the bottom, at 400bp. But there is a large smear running down and this gel looks nothing like that of the pre-cast gel. I am afraid I cannot purify this band because I lost some of the product due to smearing.

What does this smear mean? Did I overload the gel, did I run gel too fast? Should I use TAE instead of TBE? I always run with those conditions, so I didn't do anything out of my norm. But I need to purify only that band seen in the precast gel, how can I make it look the same on the poured gel?

I appreciate any advice. Thanks
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File Type: jpg gelpic2.jpg (10.7 KB, 13 views)
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Old 02-07-2012, 03:51 AM
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Default Re: need help with my gel

biochem123, how do you purified the band? Gel cut? or just PCR purification? If you are using purification kits most likely the kit will mention what rectification require if you encounter this problem.
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Old 02-07-2012, 11:39 AM
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Default Re: need help with my gel

it will be a gel cut
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Old 03-10-2012, 09:00 AM
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Default Re: need help with my gel

I think there is some degradation of your PCR product, this is one possibility. The second possibility, you may be using so much DNA for your PCR try to use no more than 30 ng of DNA for PCR, store leftover PCR products at -20 degrees and run it in lower gel percent e.g 1.5%, TAE is better than TBE for band recovery

Good Luck
Alaa
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Old 03-31-2012, 09:43 PM
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Default Re: need help with my gel

I think there was something wrong with your second gel. There's tons of DNA stuck in the well, also the ladder looks smeary. Maybe check your buffers.

Also TBE is not the best for DNA recovery from gels, TAE is preferred for this. It's something about the borate and agarose that makes it hard to solublize the DNA using your standard QIAGEN kit of whatnot.
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Old 04-25-2012, 07:07 AM
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Default Re: need help with my gel

biochem123, any update on this?
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