Rapid Isolation of Small Quantities of DNA from Physarum Amoebae

Tim Burland (Last Update September 23, 1994 )

To isolate top-quality DNA  from Physarum, use the protocol for isolation of nuclear DNA.

This rapid protocol  yields amoebal DNA that is less pure, but can be applied to fewer amoebae, such as the number that can grow on a single 9-cm plate. Whether amoebae are axenic or not, the most rapid way to obtain DNA starting from an amoebal colony on a plate (e.g. a transformant), is to grow amoebae on bacterial lawns on plates rather than axenically.

1 - Amoebae grown on bacterial lawns

• Harvest growing amoebae in water and subculture with live bacteria onto SM plates at 5x10⁵ amoebae/plate.
• Incubate 26^o for 2-3 days, until just becoming confluent (should be 1-2x10⁸ / plate), or, for strains that self rapidly, at 30°.

(Vary temperature or initial inoculum to obtain cells at the right stage of growth—you want as many active amoebae per plate as possible, with as few cysts as possible, as cysts will not lyse readily, will slowly release nucleases, and will thus degrade DNA during lysis.)

• Harvest 0.5-2x10⁸ amoebae in water, spin down 2min at 200xg in a benchtop centrifuge, and wash twice in water to remove bacteria. Resuspend cells in 10mLwater.
• Rock or shake at 20-30^o for 2-3hr—this promotes flagellate development and gets most nuclei through S phase; if time is short, you can reduce or omit this incubation in water.
• Check using a phase-contrast microscope that the ratio of amoebae:bacteria is less than 1:1 (if not, wash further in water). Spin down amoebae again and resuspend in residual liquid, then lyse (Step 3)

2 - Axenic amoebae

• Maintain culture in exponential growth.
• Harvest 0.5-2x10⁸ amoebae by centrifugation at 200xg) for 2min in a benchtop centrifuge.
• Wash once in 10ml BSS, resuspend amoebae in residual liquid, then lyse (Step 3)

3 - Lyse amoebae

• Mix lysis buffer with 1/20th vol Proteinase K stock.
• Then add 2.5 ml [lysis buffer+proteinase K] to the amoeabae and IMMEDIATELY transfer to a 70^o water-bath.
• MIX GENTLY - DO NOT VORTEX. Incubate 15min.
• Cool to 37^o, add 125ul Proteinase K, & incubate 2h to overnight @ 37^o

(If reducing Proteinase treatment to 2hr, extract with phenol and chloroform before RNase treatment)

4 - Digest RNA

Add 300ul RNase solution and incubate 37o for 2hr, or overnight if more convenient

5 - Organic Extractions

• Extract once each with an equal volume of

1. Buffer-saturated phenol,
2. A 1:1 mix of buffer-saturated phenol : chloroform/isoamyl alcohol (24/1), and
3. Chloroform/isoamyl alcohol (24/1) (all room temp).

• If the phenol-chloroform extraction leaves a large mess of material at the aqueous-organic interface, do a second phenol-chloroform extraction before the chloroform extraction.

6 - Precipitate DNA

• At room temp., add 1/15th vol 3M sodium acetate then 2 volumes of ethanol & mix gently.
• Ppte should appear in a few seconds (if it does not, try incubating at -20^o for 30-120 min)
• Retrieve ppte - If ppte is stringy (as it should be) and in one piece, pluck it out with a sterile dispo plastic pipette, or pour off soup while holding ppte in place with a pipette. Otherwise, centrifuge 2min @ full speed in a benchtop centrifuge.

7 - Wash and resuspend DNA

• Wash ppte x2 in 70% ethanol. If the pellet is in one piece, you don't need to spin between washes, you can just pour off the supernatant
• Drain well in air to dry off the ethanol and most - but not all - of the moisture (totally dry pellets take several days to redissolve)
• Resuspend DNA in 125 ul TE. Dissolution is slow - heat to 65° for an hour if in a hurry.
• Estimate DNA concentration and purity by OD_260/280nm.
• 100% yield from 10⁸ haploid amoebae would be 60ug (480ug/ml)