John M. S. Bartlett.pdf - Bio-Nica.info

30 Bartlett and White
11. 5 M sodium perchlorate: Dissolve 70 g of sodium perchlorate in 80 mL of distilled water.
Make up to 100 mL.
12. TE Buffer, pH 7.6: Take 10 mL of 1 M Tris-HCl, pH 7.6, 2 mL of 0.5 M EDTA, and make
up to 1 L with distilled water. Adjust pH to 7.6 and autoclave 15 min at 15. p.s.i.
13. Chloroform prechilled to 4°C.
14. Ethanol (100%) prechilled to 4°C.
3. Method
3.1. Blood Collection
1. Collect blood in either a heparin- or EDTA-containing Vacutainer by venipuncture (see
Note 2). Store at room temperature and extract within the same working day.
3.2. DNA Extraction
To extract DNA from cell cultures or disaggregated tissues, omit steps 1 through 3.
1. Place 3 mL of whole blood in a 15-mL falcon tube.
2. Add 12 mL of reagent A.
3. Mix on a rolling or rotating blood mixer for 4 min at room temperature.
4. Centrifuge at 3000g for 5 min at room temperature.
5. Discard supernatant without disturbing cell pellet. Remove remaining moisture by inverting
the tube and blotting onto tissue paper.
6. Add 1 mL of reagent B and vortex briefly to resuspend the cell pellet.
7. Add 250 µL of 5 M sodium perchlorate and mix by inverting tube several times.
8. Place tube in waterbath for 15 to 20 min at 65°C.
9. Allow to cool to room temperature.
10. Add 2 mL of ice-cold chloroform.
11. Mix on a rolling or rotating mixer for 30 to 60 min (see Note 3).
12. Centrifuge at 2400g for 2 min.
13. Transfer upper phase into a clean falcon tube using a sterile pipet.
14. Add 2 to 3 mL of ice-cold ethanol and invert gently to allow DNA to precipitate (see
Note 4).
15. Using a freshly prepared flamed Pasteur pipet spool the DNA onto the hooked end (see
Note 5).
16. Transfer to a 1.5-mL Eppendorf tube and allow to air dry (see Note 6).
17. Resuspend in 200 µL of TE buffer (see Notes 7 and 8).
4. Notes
1. Autoclaving sugars at high temperature can cause caramelization (browning), which
degrades the sugars.
2. As will all body fluids, blood represents a potential biohazard. Care should be taken in all
steps requiring handling of blood. If the subject is from a known high risk category (e.g.,
intravenous drug abusers) additional precautions may be required.
3. Rotation for less than 30 or over 60 min can reduce the DNA yield.
4. DNA should appear as a mucus-like strand in the solution phase.
5. Rotating the hooked end by rolling between thumb and forefinger usually works well.
If the DNA adheres to the hook, break it off into the Eppendorf and resuspend the DNA
before transferring to a fresh tube.
6. Ethanol will interfere with both measurements of DNA concentration and PCR reactions.
However, overdrying the pellet will prolong the resuspension time.