Extraction of DNA from whole blood

Background

This protocol is adopted from Bartlett, J. M. S. and D. Stirling (2003), PCR protocols, Ch 6.

Materials

  • 15 ml centrifuge tubes
  • 1.5 ml centrifuge tubes
  • Waterbath (65°C)
  • Tube rotator

Methods

Blood can be collected in heparin or EDTA-vacutainer tubes. Process samples on the same days as collection if stored at room temp.

  1. Move 3mL of whole blood to a 15 mL tube.
  2. Add 12 mL of Reagent A
  3. Mix slowly by rotation, 4 min at room temp.
  4. Centrifuge 3000 g for 5 min at room temp.
  5. Discard supernatant without disturbing cell pellet. Remove all moisture by inverting and blotting on paper or by pipetting.
  6. Add 1 mL of Reagent B, vortex to re-suspend the cell pellet.
  7. Add 250 μl of 5 M sodium perchlorate and mix by inverting tube several times. ⚠ Sodium perchlorate is harmful if swallowed and causes irritation. Keep away from heat.
  8. Place the tube in 65°C waterbath for 15-20 min.
  9. Cool to room temp. 10 Add 2 mL of ice-cold chloroform. ⚠ Toxic, perform operation in fume hood with appropriate protective equipment.
  10. Mix on a rotating mixer for 30 to 60 minutes (more or less will reduce DNA yield).
  11. Centrifuge, 2400g for 2 min. Careful not to disturb the separated phases.
  12. Transfer upper phase into a clean falcon tube with a sterile pipette.
  13. Precipitate the DNA by adding 2-3 mL of chilled 100% ethanol, invert tube. DNA should appear in the solution.
  14. Transfer the DNA to a 1.5 mL tube allow to air dry.
  15. Resuspend in 200 ul of TE buffer.
  16. Quantify DNA concentration with the spectrophotometer, 200-500 ng/ul is expected.

Solutions

0.5 M EDTA, pH 8, for 1 l:

Component Weight
EDTA 146.1 g
NaOH ~20g

Add EDTA to 800 mL ddH2O, adjust pH with NaOH. Autoclave 15 psi, 15 min.

1 M Tris-HCl, pH 7.6, for 1 L:

Component Weight
Tris-base 121.1 g

Add to 800 mL ddH2O, adjust pH with HCl and make up to 1L. Autoclave 15 psi, 15 min.

Reagent A, red blood cell lysis, pH 8. For 1L:

Component Concentration Weight/Volume
Tris-HCl 0.01 M 10 ml 1 M Tris-HCl
Sucrose 320 mM 109.54 g
MgCl2 5 mM 0.47 g
Triton X-100 1% 10 mL

Adjust pH to 8 with NaOH. Autoclave 10 psi, 10 min.

Reagent B, cell lysis

Component Concentration Weight/Volume
Tris-HCl 0.4 M 400 mL 1 M Tris-HCl
NaCl 150 mM 8.76 g
EDTA 0.06 M 120 mL 0.6 M EDTA
SDS 1% 10 g1

Adjust pH to 8 and make to 1 L with ddH2O. Autoclave at 15 min at 15 psi2.

5 M sodium perchlorate, for 100 mL:

Component Weight/Volume
sodium perchlorate 70 g

Dissolve in 80 mL ddH2O make to 100 mL.

TE-buffer, for 1 L:

Component Concentration Weight/Volume
Tris-HCl 0.01 M 10 mL 1 M Tris-HCl ph 7.6
EDTA 1 mM 2 mL 0.5 EDTA

Adjust pH to 7.6, make to 1 L, autoclave 15 min at 15 psi.

Chloroform, chill to 4°C.

Ethanol (100%), chill to 4°C.

Footnotes

  1. Add SDS after autoclaving.↩︎

  2. Add SDS after autoclaving.↩︎