GEL Extraction (any advanced techniques ?) - (Jun/11/2008 )

Hi all,

Is there any better method/technique than using the Qiagen DNA gel extraction kit ??? If so please let me know.
I just wanted to extract very very weak bands from gel.

Thank you,
Regards,

Biolearner...

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Learner, I am not aware of an advanced or better technique, but you can check out many different ones here:
http://search.vadlo.com/b/q?sn=158621799&a...tion+&rel=0
..

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If your DNA is within your gel, i dont know any method other than gel extraction
And please be noted that a very very weak bands in gel will result very very low concentration of DNA after purifying it.
by the way, what is your sample?and the length of nucleotide sequence?

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Yep its gel extraction only!
But I don’t want to lose the DNA after purifying it.
My sample is linear ligation sample. (assembly of three fragments (each 3kb))
Total length of the nucleotide sequence is about 9kb. ….
Thank you…

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Thank you, I shall go through that link...

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Hi Learner -- I've had very good results with the Qiaquick kit, and I would recommend doing the optional isopropanol step before running through the column. (check the protocol) Also, when eluting, let column sit for 1-2 minutes after adding your elution buffer (or water) before you spin. Keep your elution volume low to increase your final concentration..
Otherwise, for future gels, you can try electrophoresing until bands are resolved, then cutting bottom of gel off below your band and continuing electrophoresis to run your band out of gel onto a membrane .. then recover with water or buffer. (I haven't done this, but have seen it done) :-)

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Yeah, I was going to mention this.

Another method is to skip the membrane ... cut a wedge in the gel right ahead of your band, and as you run the band into the 'wedge' area just suck out the fluid (and DNA) every few minutes until the band is all run out. Or, cut the band, put it in a dialysis tubing bag, with running buffer, and electrophorese (the dialysis tubing bag catches the DNA as the gel band becomes 'empty' ).

All of these severely dilute your sample. You could concentrate it or precipitate it.... What's your downstream application?

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There are better ways to clone these fragments. Splice two or all of them together by splice overlap PCR or clone each fragment into the vector one at a time. Or try a 4-way ligation. A combination of these techniques can be used. This method of ligating the products together and electrophoresing is probably the worst of the options you have in my opinion, just because the yield is so low/nothing. And it's going to be virtually non-existant trying to get 3 fragments together. I would revise your strategy because i think you're really wasting time with this one. No offense intended, i just think you'll never get this one to work so you should move on to something better.

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