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Sunday, 18 November 2007

DNA isolation protocol (1)

Isolation of Genomic DNA
DNA isolation from whole blood
Materials
10 ml fresh or thawed frozen whole blood
NKM buffer, 4 degree celcius
Resuspension buffer
10 X TEN solution
2 mg/ml proteinase K
10% (w/v) SDS
Buffered phenol
25:24:1 (v/v) phenol/chloroform/isoamyl alcohol
24:1 (v/v) chloroform/isoamyl alcohol
TE buffer, pH 8.0
Dailysis buffer
Refrigerated centrifuge
Orbital shaker
Dialysis tubing (MWCO 50000)
1.5 ml storage vials

6 comments:

praspowt said...

1. Bring 1 to 15 ml fresh or thawed whole blood (scale proportionally for smaller quantities) to 40 ml with 4 degree celcius NKM buffer. Vortex, then centrifuge 30 min at 3500 X g, 4 degree celcius. Remove all but 10 ml supernatant. Resuspended pellet and vortex (and pipet up and down with a Pateur pipet if necessary) to break up any clamps.

praspowt said...

2. Add resuspension buffer to 40 ml, balance tubes, and centrifuge 30 min at 3500 X g, 4 degree celcius. Remove all but 4 ml supernatant. Add:
0.5 ml 10X TEN solution
0.25 ml 2 mg/ml proteinase K
0.5 ml 10% SDS
Mix gently and incubate overnight at 37 degree celcius to allow digestion

praspowt said...

3. Extract DNA twice with an equal volume of buffered phenol, once with an equal volume of 25:24:1 (v/v) phenol/chloroform/isoamyl alcohol, and twice with an equal volume of 24:1 chloroform/isoamyl alcohol. For each extraction: mix gently on an orbital shaker 30 min at room temperature, centrifuge 15 min at 1500 X g, room temperature, and remove and discard organic phase (usually the bottom layer; phases may invert in presence of high salt)

praspowt said...

4. Transfere DNA solution into dialysis tubing. Dialyze four times, 12 hr each time, with dialysis buffer. Save 0.3 ml of the last change of dialysis buffer. Transfer dialyzed DNA solution to 1.5 ml microcentrifuged tubes.

praspowt said...

5. Quantitate DNA by diluting 0.1 ml DNA solution in 0.2 ml TE buffer and reading the A260 and A280 of the diluted DNA (A260/A280 ratio > or equal 1.7 is usually suitable) as well as the last change of dialysis buffer and unused dialysi buffer (which should give the same absorbance readings if dialysis is complete)

praspowt said...

6. Dilute DNA appropriately with TE buffer and divide into aliquots in 1.5 ml storage vials that can be tightly sealed. Add 40 microlitre chloroform to eac aliquot to kill any contaminating microorganisms. Label tubes, seal tightly, and store up to several years at 4 degree celcius. If DNA samples are to be used for PCR only, store at -70 degree celcius until use.
Avoid freezing if DNA is to be used for Southern analysis to prevent DNA degradation during freeze-thaw cycles.