[DIYbio] Re: Your favorite ethanol precipitation protocol

Ethanol precipitation with NaCl

Materials ·

 Nucleic acid solution ·

 2M NaCl

 Isopropanol or ethanol ·

// 96% EtOH and cold 70% EtOH

 TE Buffer pH 8 or nuclease-free water

Method

1. Add 1/10 volume of 2M sodium chloride to the nucleic acid in solution. // 40 uL DNA -> 4 uL NaCl (2M)

2. Add 2.5 volumes of EtOH (or 1 volume of isopropanol). Gently mix. // 44 uL solution -> 110 uL EtOH

3. Incubate for 1 hour at -20°C or overnight. -80 is better but optional.

4. Centrifuge for 5-10 minutes at 14,000 x g (at 4°C if possible). Discard the supernatant, don't disturb the pellet.

5. Rinse the pellet with cold 70% ethanol.

6. Centrifuge for 5-15 minutes at 12,000 x g (at 4°C if possible). Discard the supernatant carefully to avoid disturbing the pellet.

8. Air dry the pellet for 5-10 minutes, being careful to not over-dry, which may render the pellet more difficult to dissolve. Note: Isopropanol may require longer drying time than ethanol.

9. Dissolve the nucleic acid pellet in nuclease-free water or TE Buffer, pH8.

See picture attached. It's Lambda-Hind; 100 bp extended ladder; only primers; PCR band that has been precipitated withthe above protocol. The PCR is a 600 bp fragment of the rbcL gene. Looks correct. 

I used very high EtOH (>96%) from a half-full flask that has been standing around for > 1 year. 

Sequencing data follows soon, then we'll have the final confirmation.