DNAExtractionfromTissue

實驗概要

DNA extraction from tissue.

主要試劑

Extraction buffer

100 mM Tris-HCl (pH 8.0)     

100 mM EDTA (pH 8.0) 

100 mM Na-Phosphate (pH 8.0)   

1.5 M NaCl

1% CTAB (Hexadecyltrimethylammonium-bromide)

SDS (10%)

Proteinase K (20 mg mL^-1 in autoclaved MilliQ and filter sterilized)

Phenol/Chloroform/Isoamylalcohol (25:24:1 w/v)

Chloroform/Isoamylalcohol (24:1 w/v)

Isopropanol p.a.

Ethanol p.a., 70%

MilliQ, autoclaved

TE-Buffer, 0.5x

5 mM Tris-HCl

0.5 mM EDTA

pH 7.0-8.

實驗步驟

1.  Add 4 ml extraction buffer and 50 μl Proteinase K [20mg/mL] to ~ 300 mg  of tissue and incubate at 37°C for 11 or 12 hr or until all tissue is  dissolved (mix the samples in between). (If the tissue is really compact  you can increase the digestion step up to an overnight incubation.).

2. Add 440 μL 20% SDS and invert samples several times.

3. Incubate samples at 55°C for 2 hr, invert samples at least every 20 min.

4. Centrifuge samples  at 4°C, 16000 xg, 30-60 min (sample dependent) (after centrifugation you  should see a white swimming pellet).

5. Transfer the liquid phase into a new tube and place it on ice.

6. Add 2 ml extraction buffer and 200 μl 20% SDS to the remaining pellet, mix and incubate 20 min at 55°C.

7. Add 1 ml Phenol:Chloroform:IAA (25:24:1), mix and incubate 15 min at 65°C.

8. Centrifuge at 16000 xg for 20 min, 4°C.

9. Transfer the liquid phase to step 5.

10. Add 1 vol  Phenol:Chloroform:IAA (25:24:1) to the combined liquid phases, invert  several times and centrifuge at 16000 xg for 20 min, 4°C.

11. Transfer the  supernatant into a new tube and add 1 vol Chloroform/ IAA (24:1), invert  several times and centrifuge at 16000 xg for 15 min, 4°C, transfer the  supernatant into a new tube.

12. Combine the  interphases from the steps 8, 10 and 11, add 1 vol Chloroform/ IAA  (24:1), invert several times and centrifuge at 16000 xg for 15 min, 4°C.

13. Transfer the supernatant to the supernatant from step 11.

14. Add 1 vol  Chloroform/ IAA, invert several times and centrifuge at 16000 xg for 15  min, 4°C, transfer the supernatant into a new tube.

15. Add 0,6 vol Isopropanol and mix well by inversion, incubate 1 h at RT or overnight at 4°C.

16. Centrifuge at 16000 xg for 45 min and discard the supernatant.

17. Add 3 ml 70% ethanol and centrifuge 15 min at 16000 xg, discard the ethanol.

18. Let the pellet air dry.

19. Dissolve the pellet in an appropriate volume of 1x TE-buffer overnight at 4°C.

注意事項

The steps 12 -14 are optional. If you’re afraid of having not enough DNA you should do them.