Gel Purification -- Anyone else have this problem?
s535290 at aix1.uottawa.ca
Fri May 23 15:53:19 EST 1997
> This is exactly why I stopped using these gel extraction kits. I
> abandoned them after I found this spin-out technique. It has worked
> well and reliably for me in the past year that I have used it.
> The reference is (biotechniques 17(4) p634 1994) Chuang and Blattner
> Ultrafast recovery of DNA by centrifugation thru a paper slurry.
> Basically, you make a paper slurry by cutting up a 10x10 piece of 3mm
> paper (like the one used to back gels for drying), place the pieces in
If you don't trust gel purification methods like geneclean or quiaex
(which seem to work rather well in my hands - I always do an extra round
of washing and let the pellet air dry much longer than is mentioned in
their instructions - I think residual wash buffer is what is killing
downstream enzymatic steps for most people) a much simpler method is the
filter tip method. Also published in biotechniques.
You cut about 0.5 cm from a P200 filter tip, such that it fits in a 1.5 mL
eppy tube. Then you squeeze the gel slice onto the top "chamber" of the
tip (ie. where the barrel of the P200 fits onto the tip), place on the
eppy tube, spin for 10' at 4000 RPM in a standard minicentrifuge. Don't
spin faster, or the agarose starts squeezing through the pores of the
filter tip. You can then ethanol precipitate the DNA, though if the gel
was destained in distilled water (or in Tris-EDTA), the DNA solution is
essentially ready to be used with any enzyme. Works like a charm, is very
cheap, and is FAST FAST FAST. Not long ago I gel purified 24 gel isolated
samples from an ExoIII deletion series in 20 minutes, and the DNA was
recircularized with ligase without any problems. DNA yield is about %50.
A few brands of filter tips were tested in the original paper, but we use
Costar filter tips and they work well.
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