gel extraction - (May/09/2008 )

Hi,
I used QIAGENE gel extraction Kit to isolate 5kb fregment, but after gel extraction, I got a 2kb fragement instead of 5kb.
so the second time, I isolate DNA fragment w/o kit but I have the same result, 5kb fragement become 2kb,(only one 2kb band)
I am very sure I started with 5kb fragement.

Any suggestions would be very appreciated.

Thanks

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It sounds like there may be several problems here. Based on the size of your fragment, I am assuming you are cutting out a vector that you digested? How much DNA are you starting with (in terms of ng)? In any case, I would try it again. Make your agarose gel 0.7-0.8% and as thin as possible, and run it very slowly (30-50V for several hours) Also, when you pour in TAE or TBE buffer into the gel box, only pour in enough to just barely cover the gel. The more buffer you pour in, the more resistance the DNA has to travel, and thus smaller bands may appear larger than they really are. You really want GOOD separation when cutting out a band. So, when I say make your gel thin, it's purpose is so that you can have a much better visual perspective on your band. If the amount of DNA you have exceeds the maximum volume you can load in your gel, then ethanol precipitate your DNA, and reconstitute it in a small volume (20-30uL) of TE buffer.

The other issue is contamination from nearby wells. Make sure you only run your product in the gel, plus a 1kb ladder. Put the ladder in the first lane, and your product 4 lanes over. Make sure your razor blade does not cut into the 1kb ladder lane. Also, before you put your gel on the UV box, wipe it down real good with water and dry it.

The final possibility is that, if you are running a gel on plasmid DNA you MUST digest it with one unique enzyme to make it linear for correct sizing. Supercoiled plasmid DNA when run directly on a gel ALWAYS appears smaller than it really is. (kind of like rear-view mirrors on a car)

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