DNA Isolation Protocols

A quick "dirty" prep is usually sufficient, while some genotyping may work better with highly purified DNA. Determine empirically which protocol works best for your genotyping.

1. NaOH extraction (quick "dirty" DNA preparation). Reference: Truett GE et al. 2000. Biotechniques 29(1):52-54

• Cut 2mm of tail and place into an Eppendorf tube or 96-well plate.
• Add 75ul 25mM NaOH / 0.2 mM EDTA.
• Place in thermocycler at 98ºC for 1 hour, then reduce the temperature to 15°C until ready to proceed to the next step.
• Add 75ul of 40 mM Tris HCl (pH 5.5).
• Centrifuge at 4000rpm for 3 minutes.
• Take an aliquot for PCR (use 2 ul undiluted, or 2 ul of a 1:100 dilution/reaction).

This protocol yields a highly purified DNA preparation from mouse tail biopsies.

1. Remove 0.5 mm of tail into polypropylene microfuge tube (do not mince). (The tubes must have tight-fitting caps, so that there are no leaks in steps 3 and 7 below.)

2. Add 0.5 ml DNA digestion buffer with proteinase K added to 0.5 mg/ml final concentration. (0.5 mg/ml is a high concentration and can probably be reduced.)

DNA digestion buffer:

• 50 mM Tris-HCl pH 8.0
• 100 mM EDTA pH 8.0
• 100 mM NaCl
• 1% SDS

3. Incubate overnight at 50-55 °C with gentle shaking. (At this step, mechanical agitation greatly aids complete disruption of the tail.)

4. Quick-spin tubes to get solution off inside of cap.

5. Fill inside well of microfuge tube cap with vacuum grease. (We use Dow Corning high-vacuum grease and a 10cc syringe to dispense.)

6. Add 0.7 ml neutralized phenol/chloroform/iso-amyl alcohol (25:24:1).

7. Mix fairly vigorously. (Do NOT vortex; we use a clinical rotator for 1 hour.)

8. Spin in microfuge at top speed 5 minutes and transfer 0.5 ml of the upper phase to new microfuge tube. (Use P1000 for transfer, and draw the aqueous phase gently through tip several times after transfer if the DNA is still in large, gelatinous mass.)

9. Add 1 ml 100% ethanol at room temperature and invert (using clinical rotator if you wish) until DNA precipitate forms. (approximately 1 minute).

10. Spin in microfuge 5 minutes and carefully remove and discard supernatant.

11. Add 0.5-1 ml 70% ethanol (-20 °C) and invert several times.

12. Spin in microfuge 5 minutes and carefully remove and discard supernatant.

13. Quick-spin tubes and remove last drop of ethanol solution with 25 µl capillary tube.

14. Air dry at room temperature or in dessicator (overnight if you wish).

15. Add 100-200 µl TE buffer and incubate at 65 °C for 15 minutes to resuspend DNA. Draw DNA through P1000 tip after 65 °C incubation to aid in suspension if you wish.

16. Use 10-20 µl for restriction enzyme digest.

17. Total yield is approximately 20-50 µg DNA, 0.1-0.25 µg/µl.

1. Obtain 65-100 µl of blood by retro-orbital bleed with a heparinized microcapillary tube. Expel blood immediately into a 1.5 ml microfuge tube containing 20 µl of 10 mM EDTA. Mix immediately to prevent clot formation. Store on ice until processing.

2. Add 200 µl lysis buffer to each tube and vortex to suspend evenly.

3. Microfuge 25 seconds at 16000xg to pellet nuclei.

4. Remove and discard supernatant and repeat steps 2-4 two more times, or until no hemoglobin remains.

5. Resuspend nuclear pellet in 100 µl PBND with 60 µg/ml proteinase K and incubate at 55 C for 60 minutes (or overnight, if convenient).

6. Heat samples to 97 C for 10 minutes to inactivate proteinase K.

7. Add 1-5 µl of DNA solution for a 25 µl PCR reaction.

Reagents:

1) Lysis buffer

• 0.32 M sucrose
• 10mM Tris-HCl (pH 7.5)
• 5 mM MgCl2
• 1% v/v Triton X-100

2) PBND (PCR buffer with nonionic detergents)*

• 50 mM KCl
• 10 mM Tris-HCl (pH 8.3)
• 2.5 mM MgCl2
• 0.1 mg/ml gelatin
• 0.45% (v/v) Nonidet P40
• 0.45% (v/v) Tween 20
• Autoclave to sterilize and dissolve gelatin.
• Store frozen.

*Add proteinase K (60 µg/ml) immediately prior to use).

(Adapted from Higuchi, R. (1989) Amplifications 2: 1-3)