A cheap DNA extraction protocol.

1. Make sure there is ethanol in the freezer.
2. Place in a 1.5ml eppendorf tube 200 μl Digsol buffer and 10 μl Proteinase K (10 mg/ml).
3. Remove fly from ethanol (with forceps) and blot onto tissue. When dry, transfer to the eppendorf.
4. Disrupt the fly with electric pestle machine.
5. Wrap the eppendorf rack in tissue paper and elastic bands and place in rotating oven at 55 oC (3hrs) or 50 oC (overnight).
6. (Optional) Add 3 μl 10 μg/μl RNase A¹ and incubate at 37oC for 45 min.
7. Add 300 μl 4M ammonium acetate to each sample.
8. Vortex several times over a period of at least 15 minutes at room temperature to precipitate the proteins.
9. Centrifuge² for 10 minutes at 13,000 rpm ³. Label new eppendorfs.
10. Aspirate 500 μl supernatant into the clean labelled 1.5ml eppendorfs⁴.
11. Add 1ml 100% ethanol and mix by inverting. The DNA should precipitate out of solution, but may not be visible from single flies, especially males.
12. Leave samples in freezer at -20 oC for 1h.
13. Centrifuge for 10 minutes at 13,000 rpm.
14. Pour off ethanol in a smooth movement so as not to lose DNA pellet.
15. Add 500 μl 70% ethanol and invert several times to rinse pellet.
16. Centrifuge for 5 minutes at 13,000 rpm⁵.
17. Pour off ethanol in a smooth movement and stand tubes upside-down on clean tissue until they are completely dry (1 h or overnight incubation).
18. Once fully dry add Low TE. The amount added is dependent on pellet size, 20 μl results in up to 50 ng/ul DNA from a single female D. pseudoobscura.
19. Place tubes in shaking incubator for 30 minutes (55 oC) to dissolve pellet (flicking every 10 mins). Or leave overnight at room temperature. You are aiming for a fully homogeneous, slightly viscous solution.
20. Store at -20 oC (long term) or 4 oC (short term).

Recipes

1M Tris-Base (MW 121.1) pH 8.0

(Tris Hydroxymethyl Aminomethane)

For 200ml:

• Dissolve 24.22g in distilled water by stirring
• pH should be about 8.0
• Autoclave to sterilise

0.5M EDTA (MW 372.2) pH 8.0

(EthyleneDiamineTetraacetic Acid)

For 200ml:

• Dissolve 37.2g in distilled water by stirring.
• Will need to pH solution with NaOH whilst it is dissolving (in order for all EDTA to solubilise).

20% SDS

(Sodium Dodecyl Sulphate)

For 100ml:

• Add 20 g SDS (use autoclaved water as end solution cannot be autoclaved).
• Use a fume hood and wear a mask when weighing this powder.
• It is easier to dissolve by heating at 55 oC than by shaking.

Digsol pH 8.0

(Digestion Solution - Bill Amos and Josephine Pemberton)

| Final Concentraction | Stock | For 1000ml | For 200ml | | -------------------- | ------------------- | ---------- | --------- | | 20mM EDTA | EDTA (0.5M, pH 8.0) | 40ml | 8ml | | 120mM NaCl | NaCl | 6.85g | 1.37g | 50mM Tris | Tris (1M, pH 8.0) | 50ml | 10ml | | | Distilled water | 810ml | 172ml | SDS (0.5%) | SDS (20%) | 25ml | 5ml | [Digsol recipe]

• Warm all constituents except SDS until dissolved
• Autoclave to sterilise
• Add SDS
• pH with HCl if necessary

4M Ammonium Acetate, pH 7.5

For 100ml

• Dissolve 30.83g Ammonium acetate⁶ in distilled water.
• Autoclave to sterilise.
• If necessary pH with Glacial acetic acid (was not necessary).

T.E. pH 7.5-8.0

| Final Concentration | For 400ml | | --------------------- | ----------- | | 10mM Tris | 4ml of 1M Tris (pH 8.5) | | 1mM EDTA | 800 μl of 0.5M EDTA (pH 8.5) | [TE recipe]

Low EDTA T.E. Buffer pH 7.5-8.0

For resuspending DNA which will be used in PCR

| Final Concentration | For 400ml | | --------------------- | ----------- | | 10 mM Tris | 4 ml of 1 M Tris (pH 8.5) | | 0.1 mM EDTA | 80 μl of 0.5 M EDTA (pH 8.5) | [Low TE recipe]

• pH if necessary
• Autoclave to sterilise

10 mg/ml Proteinase K

In 1 ml aliquots in -20 oC freezer.

In St Andrews it is kept in special solution at 4 oC at 20 mg/ml.