DNA stuck in wells - (Apr/04/2007 )

Hi,

I'm having trouble with my DNA gel right now. I loaded samples from a digest of plasmid DNA after miniprep and all of them are stuck in the wells and hardly ran through the gel. The marker and a control plasmid (that hasn't been digested) ran fine.

Any help? What can cause this kind of problem?

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Did you spot a lane with your plasmid that was uncut as well?

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Some restriction enzymes will bind to DNA and inhibit or slow down gel migration. You might try heat killing the enzymes prior to running the gels. Also, very long fragments will not run, so if you have created fragments with long single stranded overlaps which bind to each other, these fragments, despite not being ligated, may not run as single fragments on the gel.
As a blunder-stopper, you might also want to run the plasmid in restriction enzyme buffer, without enzyme (make up a master mix of DNA + RE buffer, aliquot and add enzyme or not, incubate, and run).

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one more factor I would add is over use of BSA and restriction enzyme. If you use too much BSA or enzyme, then the same would happen. The DNA gets stuck in the well

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Well I've been there. I think you've precipitated your DNA with ethanol and then overdried it before resuspending.

You can still recover the stucked DNA using gel extraction kit then run it again.

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Thanks a lot for all these comments! I really appreciate it!

@ chick gene: you might be right, I might have overdried my DNA. But if so, why does it get stuck in the wells? Is it because the DNA pellet wasn't completely dissolved after the EtOH precip and thus, I got clumps of DNA in my digest? Then it would be just be too much DNA, right?

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I don't really agree with the overdrying of DNA pellet. Most of the time I just left my DNA to dry for quite a long time (say about 30 - 45 minutes), however, my DNA pellet still dissolves well and I never have my DNA sample stucks in the well.
As Perneseblue suggested, you may have overused BSA and your enzyme. May I know how much RE did you used?

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I added 0.5µl enzyme (5U) and 2µg BSA in a 10µ reaction :-)

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2 ul BSA 10X or 100X??? (Most of the BSA come as a 100X solution).
Add SDS to the load buffer, it really help!!!!

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0.5 ul of enzyme in a 10 ul reaction could be too much. I never bother exactly how many unit of enzyme I loaded into the reaction. I always add 1 ul of the enzyme into 50 ul reaction. That's more than enough.
Good luck.

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