Silica/GuSCN extraction of RNA/DNA

Dr. J.P. Clewley jclewley at crc.ac.uk
Thu Nov 11 16:14:32 EST 1993



I was requested to post the refs about the Boom method for purifying
DNA or RNA. Here goes:
 
Silica/GuSCN extraction method:
 
Rapid & simple method for purification of nucleic acids. Boom et al, 
J Clin Micro 28: 495-503, 1990.
 
Rapid purification of hepatitis B virus DNA from serum. Boom et al, 
J Clin Micro 29: 1804-1811, 1991. (This one adds a proteinase K digest 
step to improve recovery of DNA with covalently bound protein - altho 
I have not found it necessary for good recovery of picornavirus RNA which 
has a 5 prime covalently linked protein.)
 
An inexpensive and simple method for DNA purifications on silica particles, 
Carter & Milton, NAR 21: 1044, 1993.
 
DNA extraction from Pleistocene bones by a silica-based purification method.
 Hoss & Paabo NAR  21: 3913-3914, 1993
 
VRD lab method for prep of DNA or RNA for PCR
 
Sample preparation is carried out in a laboratory set aside solely for nucleic
acid extraction for PCR. If the sample is tissue then homogenize it in TE buffer
 to give a 10% suspension. 
 
1) Add 50 ul of specimen to 900 ul lysis buffer L6 and 40 ul silica in a screw 
capped microfuge tube. Vortex the silica suspension before pipetting it. Use 
positive displacement pipettes or tips with filter plugs.
2) Vortex for 5 seconds, stand at room temperature for 5 minutes, vortex for
 5 seconds, spin for 15 seconds in a microfuge, remove supernatant with a 
plastic, fine tipped disposable pasteur pipette (pastette). Dispose into 
10M NaOH in a fume hood to avoid production of HCN.
3) Add 1 ml buffer L2 and vortex. Spin for 15 seconds and remove supernatant
 as above.
4) Repeat step 3.
5) Repeat step 3 with 70% ethanol, twice.
6) Repeat step 3 with acetone, once.
7) Dry for 10 minutes at 56 C on a dry heating block, with the lid of the tube
removed. (We have a perspex cover that sits over the entire heating block to 
protect the open tubes from contamination.)
8) Add 50 ul TE, cap the tube and vortex. Incubate at 56 C for 10 minutes,
vortex and spin for 2 minutes.
9) Collect the supernatant taking care not to carryover any silica.
 
Needs:
Screw capped 1.5 ml microfuge tubes (Sarstedt) 
 
Lysis buffer L6: Add 120g guanidinium thiocyanate (Fluka) to 100 ml 
0.1M Tris-HCl pH 6.4 and 22 ml of 0.2M EDTA pH 8.0 and 2.6g Triton X-100.
Stir overnight in the dark to dissolve. Store in the dark and use within 1 month.
 
L2 buffer: Add 120g guanidinium thiocyanate to 100 ml 0.1 Tris-HCl pH 6.4.
Heat with shaking or stirring at 60-65 C to dissolve. Alternatively, stir overnight
in the dark.
 
Silica suspension: Add 60g of silicon dioxide (Sigma) to 500 ml deionised H2O
in a measuring cylinder. Stand at room temperature for 24 hours. Remove about
430 ml of the supernatant by vacuum suction or pipetting and resuspend the 
remainder in 500 ml deionised H2O. Stand for 5 hours at room temperature.
Remove about 440 ml of the supernatant. Add 600 ul concentrated HCl to pH 2.0.
Aliquot, autoclave and store in the dark.
 
Jon Clewley
Virus Reference Division, CPHL, London NW9 5HT, UK
fax: +44 81 200 7874
 
jclewley at crc.ac.ukzzz



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