Preparation Of Control Dna From Peripheral Blood

Normal whole peripheral blood is lysed to remove erythrocytes. The remaining white cells are digested with proteinase K. The DNA is extracted with phenol and isolated.

Control DNA of mice may be prepared by applying the tumor DNA preparation protocol (see Basic Protocol 1 or Alternate Protocol 1) to the spleen or liver tissue of a wild-type mouse. Some investigators use DNA from tail preparations, but these may not be pure enough in all cases and can be resistant to digestion by DNase I during nick translation.

CAUTION: When working with human blood, cells, or infectious agents, biosafety practices should be followed; see unit 5.1.

Materials

Normal whole blood Lysis buffer (see recipe) SE buffer (see recipe) 20 mg/ml proteinase K 20% (w/v) SDS Phenol

24:1 (v/v) chloroform/isoamyl alcohol

3 M sodium acetate, pH 5.2 (see recipe in unit 8.3)

Isopropanol

70% ethanol

50-ml blood collection tube containing EDTA, heparin, or sodium citrate anticoagulant 37°C water bath

1.5-ml microcentrifuge tubes (e.g., Eppendorf) Speedvac evaporator (Savant)

Additional materials and equipment for assessment of DNA concentration (unit 4.5) and 1% agarose gel electrophoresis (unit 8.3)

Draw, lyse, and process whole blood

1. Draw 10 ml whole blood into collection tubes containing EDTA, heparin, or sodium citrate as anticoagulant, transfer into 50-ml tubes, and add 30 ml lysis buffer. Shake gently.

2. Incubate 30 min on ice.

3. Centrifuge 10 min at 300 x g, 4°C and remove supernatant (blood waste).

4. Add 10 ml lysis buffer and resuspend the pellet.

5. Centrifuge 10 min at 300 x g, 4°C and remove supernatant (blood waste).

6. Add 5 ml SE buffer and resuspend the pellet.

7. Centrifuge 10 min at 300 x g, 4°C and remove supernatant (blood waste).

8. Add 5 ml SE buffer and resuspend the pellet.

Digest cell pellet

9. Add 250 |l of 20 mg/ml proteinase K and 250 |l of 20% SDS. Shake gently. 10. Incubate overnight in a 37°C water bath.

Extract DNA with phenol

11. Add 4.5 ml SE buffer and 10 ml phenol. Mix by inverting the tube for 10 min.

12. Centrifuge 5 min at 1500 x g, 10°C. Transfer the supernatant into a new 50-ml centrifuge tube.

Molecular Cytogenetics

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