Genomic DNA isolation kits? - (Oct/21/2005 )

Hi,

So I have been trying some of the "faster" genomic DNA isolation kits, such as the promega sv genomic kit, but I have not been having much luck with getting it to work with my cell samples, ie: ductal epithelial cells.

Can anyone recommend a kit which has worked consistantly?

I use a homebrew method for isolation of genomic DNA from anything in about an hour or two; I got it at

www.protocol-online.org

it stays stable a long time and works just fine in PCR if you do an additional ETOH purification. i have used it for both human cells and bacterial cells and had all sorts of luck.

The following method doesn't require any kit and gives very consistant results:



gDNA ISOLATION FROM TISSUE CULTURE CELLS WITH PROT. K



Don’t vortex gDNA too rigorously


-Lyse cells or frozen cell pellet in lysis mix, supplemented with prot.K; use 1 ml per confluent 10 cm dish (in 2 ml eppendorf tube)

-Rotate o/n @ 55°C

-Strongly recommended: add same volume of phenol/chloroform/isoamylalcohol, shake well for 30 sec. and spin for 10 min; repeat once or twice when necessary, until interphase has disappeared

- Strongly recommended: after ph/chl/iaa, perform pure chloroform treatment (1:1) to get rid of phenol traces; proceed with upper phase

-Precipitate gDNA by adding equal volume of isopropanol, mix well by inversion (or: use 20 ml mussel glycogen -10 mg/ml stock prepared in mQ- and EtOH precipitate)

-Fish out gDNA with pipettip (or: spin briefly 5K and remove sup)

-Transfer gDNA through eppendorf tube containing 70% ethanol (wash samples separately to avoid contamination)

-Dissolve gDNA in T10E0.1, use 0.4 ml per confluent 10 cm dish (0.1 ml for small pellets); rotate gDNA for 3 hrs to o/n (@37ºC)

-Determine DNA/protein concentration @260/280 nm



Lysis mix (optional: make stock without prot.K for storage @RT)

0.1M TRIS pH 8.5
0.2 M NaCl
5 mM EDTA
0.2% SDS

Add FRESH prot.K to final conc. 100 µg/ml (from 20 mg/ml stock prepared in water, stored @ -20°C)

Thanks Theo,

Can the cell lysate from your above protocol be stored @-20 or -80C untill being processed without noticable problems? The reason why I ask is we obtain 3-4 samples a week on various days and I usually wait till the end of the week to do isolations.

My starting cell concentrations range from 100,000 to 1 million ductal epithelial cells per sample. Would this method still be a viable option with a fairly low expected DNA yield even with the addition of glycogen?

Lastly, how long of a incubation step is the 55C shaker part and are all spins done at 4C/5000G?




CW