re guanidium isothiocyanate method of single-fly preps.

Eric L. Cabot elmo at helix.nih.gov
Wed Oct 12 14:19:38 EST 1994


I "wonder" why the previous message in this thread was posted in a 
platform & application5 specific manner (i.e., Macintosh/MS-Word)
since, after all, the contents are plain old text.

Anyway, here's the contents, un-binhexed, unattached, and unabashed.

Eric_Cabot at nih.gov

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Simple extraction protocol for DNA using salt without Proteinase K

GIT cell lysis and homogenization buffer.


4.0 M guanidinium thiocyanate
0.1 M Tris-HCl (pH 7.5)
1% b-mercaptoethanol
10 mM EDTA
Dissolve 50g of guanidinium thiocyanate in 10 ml of 1.0 M Tris-
HCl (pH 7.5) 0.1 M EDTA and add ddH2O to 100ml filter through 
Whatman No. 1 and store at room temperature. Aliquot as needed 
adding b-mercaptoethanol to 1%. (Sambrook et al, Molecular 
Cloning: a laboratory manual Cold Spring Harbour Press 
1989:pp.7.19)
I have found no problem in adding the b-mercaptoethanol to the 
primary solution and storing at 4oC for 3 months.

Extraction protocol

1.Place fly or bees thorax in a 1.5ml eppindorf tube with 350ml 
of GIT buffer and grind well the solution should turn a pale 
yellow and become viscous allow to lyse for 5-15 minutes on ice.

2.Add 1/2 volume (200ml ) of cold 7.5M ammonium acetate stored 
at -20oC invert 10 times place on dry ice for 5minutes or at -70oC 
and centrifuge (preferably cold) at 6000 rpm for 10 minutes to 
sediment any particular matter.

3. Pipette supernatant (400ml) into a new tube or remove the 
debris from beneath the supernatant with a wide bore pipette.

4. Add 200ml phenol invert gently 50 times, centifuge 13000 rpm 
10 minutes a pellet should be evident suck it out with a fine bore 
pipette and discard it.

5. Add 200ml chloroform/isoamyl alcohol invert gently 50 times, 
centifuge 13000 rpm for 10 minutes remove upper aqueous layer 
to a new tube (you can pipette out the lower organic phase 
instead, saves you using so many tubes).

6. Add 200ml chloroform invert gently 50 times, centifuge 13000 
rpm for 10 minutes remove upper aqueous layer to a new tube.

7. Add 2 volumes (1000ml )of ice cold ethonol invert 10 times and 
place on dry ice for 5minutes (-70oC for 15minutes).

8. Precipatate the DNA at 13000-16000 rpm (preferably cold) for 
5-10 mintes. The supernatant is poured off and any excess is 
pipetted out.

9. The pellet is washed with 200ml of 70% ethanol and spun down 
at 13000-16000 rpm for five minutes.

10. The ethanol is pipetted off with a fine bore pipette tip and 
spun once more (briefly) to remove any ethanol adherent to the 
side of the tube this is again pipetted off and the pellet is 
suspended in 100ml  of 1.0xTE 

NB There is no need to dry the pellet before resuspension and it 
goes into solution more easily if it is not dessicated, resuspension 
is aided by a couple of gently flicks do not vortex the tubes.




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