DNA EXTRACTION FROM MOUTH SWABS
Flint Group Protocols
(2) DNA EXTRACTION FROM MOUTH SWABS (for one swab in 1.3ml tube)
REAGENTS REQUIRED:
1.
Transport buffer: ( 100mM NaCl , 10 mm Tris, 25 mM EDTA, 0.5% SDS, pH=8.5)
20 ml NaCl 5M
10 ml Tris 1M (pH=7.5, 8)
50 ml EDTA 0.5M (pH=8.5)
Make up to 1 litre with distilled-deionized water (DDW)
Autoclave and add 25 ml SDS 20%
2.
Guanidine hydrochloride 6M (GuHcl, MW=95.53): 286.59g in 500 ml DDW
3.
Ammonium acetate 7.5M (NH4Acetate MW=77.08): 289.05g in 500 ml DDW
4.
Proteinase K (100mg/ml)
5.
Chloroform
6.
Nucleon resin
7.
100% cold Ethanol
8.
70% cold Ethanol
9.
TE
PROTOCOL:
1.
Add 500
l transport buffer to tubes containing the brushes with shortened handle.
2.
Add 10µl of 100 mg/ml Proteinase K, vortex and incubate at 37°C overnight (or at 65°C for 2-3hr).
3.
Add 100
l GuHCl (6M) + 50
l NH4Acetate (7.5M), vortex and incubate at 60°C for 1hr.
4.
Spin down briefly, take out the brushes and placed them in a spin baskets inside a marked 1.5ml
Tube (Eppendorf tubes). Spin down for 1 min. Discard brushes, but save baskets for reuse. Transfer liquid form 1.3ml tubes to 1.5 ml tubes.
5.
Add 600
l of cold Chloroform and mix by inverting the tubes at least 20 times.
6.
Spin down, add 75µl Nucleon resin and without re-mixing the phases, centrifuge at 11,000 rpm for
5 min.
7.
Holding the tube vertically, without disturbing the resin layer, transfer the upper phase (~600µl) into a new labelled 1.5ml tube.
8.
Spin again at 11,000 rpm for 5 min to pellet any remaining resin and transfer the supernatant to a labelled 15ml tube.
9.
Add 2 volumes of cold absolute Ethanol (~1.2ml), leave at -20°C overnight.
10.
Spin at 2500rpm for 30 min at 4°C to pellet the DNA and discard the supernatant carefully without disturbing the pellet.
11.
Wash the pellet twice: add 2ml cold 70% Ethanol and mix briefly. Re-centrifuge at 2500 rpm for 15 min at 4°C and discard the supernatant.
12.
Invert the tube on a paper towel for several minutes to remove the remaining ethanol, and air dry the pellet (beware not to dry the DNA excessively).
13.
Resuspend in 100µl DDW, mix briefly to wash the tube, and leave over night at 4 °C.
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