Fwd: Re: Genomic DNA extraction

Byron Williams byron_w14850 at yahoo.com
Mon Aug 5 10:07:55 EST 2002


Dear Andrea,

We used to do it Graham's way (I believe it is based on procedure from
Gerry Rubin's lab from the mid 80's) and it works, but a better way is
to use a kit called "Puregene DNA isolation kit" available from Gentra
systems (1-800-866-3039). It's pricey but avoid a lot of problems that
can arise from the Rubin method such as in PCR and genomic Southerns. 
I never got genomic Southerns to work very well with the Rubin
procedure.

Another way that works great for Southerns is CsCl gradient
centrifugation to obtain genomic DNA. Here it is:
Large Scale Purification of Whole Genomic DNA

This method gives a lot of DNA which digests much better and gives
better looking Southerns than the small scale fast prep. It also takes
several days and requires many flies to start with. About 5 bottles
worth should do it.

1. Add frozen flies (approx 1 gram or 5 ml) to 20 ml nuclear isolation
buffer (NIB) in glass mortar for motor-driven teflon homogenizer and
homozenize using 10-20 passes at moderately fast drive speed.
2. Transfer homogenate to 50 ml conical polypropylene centrifuge tube.
[Use disposable one-use tubes to avoid previous plasmid contamination
in Oak Ridge tubes.] Rinse glass mortar with 10 ml NIB and add to
centrifuge tube.  Spin for 15 seconds (on to off time) in Sorvall to
pellet macroscopic material.
3. Transfer supernatant to another 50 ml centrifuge tube.  
4. Spin at 5 K for 7.5 min at 4 C to pellet nuclei. Discard the
supernatant and resuspend pellet in 2 ml fresh NIB with vigorous
vortexing. After pellet is completely resuspended, add 28 ml NIB to
make up to 30 ml total.
5. Respin and resuspend in 7 ml NIB.
6.  Transfer to a 15 ml Sarstedt tube and add 2 ml of 10% sarkosyl
(N-laurysarcosine). Mix thoroughly by gently rocking capped tube until
homogeneous (for 5-10 min). Do not vortex! Stand on ice for 5-10 min.
7. Add 12 grams of CsCl (ground up) to tube. Gently mix to ensure
dissolution of CsCl and bring to 13.5 ml (using markings on side of
tube) with 2% sarkosyl in NIB. Add solution to 5/8" x 3" Beckman Quick
Seal Polyallomer centrifuge tubes. Centrifuge for 36 hr at 40K in a
Ti50 fixed angle rotor at 15 C.
8. Drip gradients by piercing a vent hole in the top of the tube with
a 25g needle and drain hole at the bottom with an 18g needle. Collect
the viscous fraction (1-1.5ml).
9. Dialyze against 3 changes of TE (4 l each time) at 4C. Anticipate
yields on the order of 100 micrograms of DNA per gram of starting
material.

NIB
10mM Tris, 60mM NaCL, 10 mM EDTA, 0.15 mM spermine, 0.15 mM spermidine
and 0.5%triton; pH 7.4
500 ml: 
10 ml 1M Tris pH 7.5
6 ml 5 M NaCl
10 ml 0.5M EDTA, pH 7.5
750 ul 100 mM spermine
750 ul 100mM spermidine
2.5 mL 100% triton-X
470 ml dH20
Autoclave.

Hope this works,

Byron Williams
bw28 at cornell.edu
http://www.people.cornell.edu/pages/bw28




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