Genomic DNA in small quants. - A summary.

PC386 at IL.AC.TAU.VE PC386 at IL.AC.TAU.VE
Wed Jun 24 12:28:33 EST 1992


Hello Dear Netmates.

Some time (quite long..) ago, I have posted a request for
protocols on isolation of genomic DNA. I have received some
answers W/ references. I'd like to repost the ref's W/ some
detailes.  Beg pardon for the great delay in reposting, but
you know- problems, problems, problems...

1:  (Received W/ thanks from Eric Atkinson).
Kim and Smithies, Nucleic Acids Research, vol. 16, no. 18,
     pp 8887-8903 (1988).  s.a. -  Joyner et al. Nature,
     vol. 338, pp 153-156, (1989)).
This method is based on a hypotonic lysis step followed by
a Proteinase K step (freez/thaw cycle here is optional). The
supernatant goes directly to PCR. The method suits a small number
of cells. I have tryed it with success. However,I got some non
specific bands (I probably used too many cells).

2:   This is from Jeff Ross - Thanks Jeff.
Nucleic Acids Res., 19:6053  . This technique is for DNA
isolation from small tissue samples.  While the protocol calls
for spooling the DNA onto a plastic pipette tip, centrifugation
would work for collecting amounts too small to spool.  They have
used this technique with samples smaller than 200 mg, including
liver, lung, peripheral blood lymphocytes, C3H10T1/2.
I haven't tryed this method yet, but intend too.

3:   Frank Chiafari sent me a very detailed protocol :-)
 essentially for suspended cells.
The full protocol can be obtained by request. The outlines are:
a. Transfer cells into 15ml tube. Add Lysing Solution. Mix and
Centerfuge . Pour off solution, drain leaving pellet.
b. Resuspend pellet in ACE Shocking Solution, transfer to labelled microfuge    g
tube; Spin ; Pour off solution, saving  pellet; Repeat ACE wash.
c. Add 300ul of Nuclei Lysis Buffer to each pellet.
d. Add  SDS, Proteinase K and RNase A , vortex ; incubate for
1hr. at 60*C; vortex again and incubate (same).
e. Add Precipitating Solution. Shake . Let stand at room temp for
3 min. Spin in microfuge.
f. Transfer supernatant containing DNA to new tube; add EtOH.
Mix until DNA comes out of solution. Spin tube ; Wash pellet with
EtOH and respin; rotovac untill barely dry; add TE & Incubate at
60*C from two hrs. to o/n.

Frank writes that the DNA is easily restriction cut and ligated. DNA isolated
from blood to be used with PCR will amplify, but will do better if chelexed
first.

4. I myself, also found a convinient way of isolation W/ a kit
(yes friends, I know, I have read the writings on the walls
regarding your opinion on the use of kits). The kit is "Gnome"
from BIO-101. I downscaled the whole procedure 1:10, and I spin
the DNA down instead of spooling. It works O.K. in my hands.


Regarding the use of Chelex- Many people have suggested it's use
in purifying genomic DNA for PCR. Chelex 100  resin is claimed to
enhance PCR's specificity and efficiency by at least 10 times.
This is produced by Bio-Rad, Cat. # 143-2832 Biotech. Grade.
Regretfully, I haven't tryed it out in my hands yet, :-(
since the stuff has to be imported to Israel which takes a month
(Not to mention about 50% added to the price).

Life's not that simple as you can see. We in Israel, have added
another law to Murfey's : "Whenever you'll need a material or
a piece of equipment BADLY and URGENTLY, it will not be in stock
- and it will take it months to get there..."

I once again want to thank all the people who spent their
precious time and effort and wrote, and apologies for the late
summary.

Be Well. Sincerely, Benny.

 **************************************************************
* Benny Shomer                                                 *
* Tel-Aviv University                                          *
* Sackler School of Medicine, Dept.of Embryology and Teratology*
*--------------------------------------------------------------*
* Snail:  Sheba Medical Center, Tel-Hashomer, 52625,   Israel. *
* E-mail :  PC386 at VE.TAU.AC.IL                                 *
* Tel :  972-3-640-9238     FAX :   972-3-642-2046             *
*                                   (c/o Dr. R. Ehrlich)       *
*                                                              *
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