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Gel forms during lysis pre DNA extraction

Gel forms during lysis pre DNA extraction - DNA Extraction Forum

Gel forms during lysis pre DNA extraction - DNA Isolation Forum. A forum specifically about questions on DNA extraction and purification.


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Old 06-22-2010, 11:25 PM
Pipette Filler
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Default Gel forms during lysis pre DNA extraction



I'm attempting to perform a DNA extraction on human lymphocytes but when I add the lysis buffer the cells form a viscous mass which is impossible to pipette. The biggest problem is that I have to perform this extraction using a lot of cells, about 15,000,000, with only 200uL of lysis buffer. I would perform this in smaller batches or with more lysis buffer but this concentration is needed for downstream tests.

I've made a variation of RIPA lysis buffer it consists of:
25mM Tris•HCl, pH 7.6
150mM NaCl
1% Igepal CA-630 (an NP-40 replacement)
1% sodium deoxycholate
0.5M EDTA

I can change this formulation as needed so long as the end result is a less viscous more pipetteable solution that won't destroy the DNA. I've already tried to modify the sodium deoxycholate concentration but the solution becomes more viscous with increased sodium deoxycholate and lower concentrations don't seem to be lysing the cells enough.

Any suggestions would be appreciated. Thanks!
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Old 07-13-2010, 02:03 PM
Pipette Filler
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Default Re: Gel forms during lysis pre DNA extraction

This may be not the solution you want, but why don't you try any extraction kit like Qiagen DNeasy blood& tissue kit. There is the handling of greater cell amounts (by the way 15*10^6 isn't such a great amount) more easy and DNA can be reconcentrated through column purifying. In Addition the purity of the extracted DNA is usually greater than by self extraction methods.
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dna , extraction , forms , gel , gel clumps , lysis , pre , ripa


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