Cleaning DNA & RNA

From Protists

Contents 1 Phenol/Chloroform (removes protein/removes phenol) 2 100% Ethanol (precipitates DNA) 3 70% Ethanol (washes out salt) 4 General Notes 5 Solution Preparation Notes

Phenol/Chloroform (removes protein/removes phenol)

1. add equal volume Phenol/Chloroform/Isoamylethanol (25:24:1; pH 6.7/8.0)

2. vortex vigorously for 30s

3. spin 5min at maximum rpm 4°C

4. transfer supernatant to a fresh tube (avoid aspiration of the interlayer or organic phase)

100% Ethanol (precipitates DNA)

1. add 0.1 volume 3M sodium acetate buffer, pH 5.2 (store at 4°C)

2. add 2.5 volumes 95-100% cold ethanol (store at -20°C)

3. vortex briefly

4. precipitate at:

• • -20°C overnight (+++)
  • -80°C 1 hour (++)
  • dry ice 15min (+)

5. spin 20min at maximum rpm 4°C

6. carefully pipet out / pour out / aspirate supernatant (do not lose DNA pellet)

70% Ethanol (washes out salt)

1. carefully add 1mL cold 70%* ethanol (store at -20°C; DO NOT vortex)

2. spin 10min at maximum rpm 4°C

3. carefully pipet out / pour out / aspirate supernatant (do not lose DNA pellet)

4. air dry 10min at room temperature with tube upside-down and open on kimwipe (do not overdry, because DNA becomes hard to dissolve)

5. dissolve in:

• • 10mM Tris pH 7.5 (+++)
  • TE-buffer (++) – EDTA may inhibit downstream enzymatic reactions
  • HPLC water (+) – freeze at -20˚C because unbuffered DNA undergoes degradation

General Notes

• Do not store RNA in water. Store RNA in (95-100%) ethanol at -20˚C.
• For immediate uses (e.g. sequencing), do not use TE buffer for storage of DNA. Use HPLC water.
• For long-term storage use TE buffer for storage of DNA. It preserves DNA by inhibiting its degradation.
• To merely concentrate clean DNA, only perform 100% & 70% ethanol steps and re-dissolve DNA/RNA in an appropriately smaller volume.
• Always use HPLC water over lesser quality water types.

Solution Preparation Notes

• To make 15mL of 3M sodium acetate:
  • measure out 6.1234g of sodium acetate trihydrate (FW 136.08)
  • pour this into a “fix” graduated cylinder and rinse off any remaining crystals with a little sterilized HPLC water into the graduated cylinder
  • fill the graduated cylinder up to 10 mL with sterilized HPLC water
  • cover very well with parafilm & shake the solution until all crystals are dissolved
  • use the pH meter (and HCL/NaOH) to bring solution to pH 5.2
  • add HPLC water up to 15mL and double check pH (& adjust if necessary)

• To make 70% ethanol, 1st fill one centrifuge tube with 100% (200 proof) molecular grade ethanol (top shelf of flammables cabinet with red cap), then get a new 15mL centrifuge tube and put 9.8mL of the 100% ethanol and 4.2mL of sterilized HPLC water into the new tube to make a total volume of 14mL of 70% ethanol final concentration.