Your favorite ethanol precipitation protocol

[Andreas "Mega" Stuermer]

Hi!

I am looking on ethanol precipitation protocols... But they all use NaOAc (which I don't have). Some indicate that you might use NaCl as well (which I have on stock). Does anyone have a working protocol how to do an ethanol precipitation with NaCl?

[Dakota Hamill]

Mix up some vinegar and baking soda, boil off water, crystalize - boom sodium acetate 

On Thu, May 9, 2019 at 10:13 AM Andreas "Mega" Stuermer <andreas.t...@gmail.com> wrote:

Hi!

I am looking on ethanol precipitation protocols... But they all use NaOAc (which I don't have). Some indicate that you might use NaCl as well (which I have on stock). Does anyone have a working protocol how to do an ethanol precipitation with NaCl?

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[Ting Kwok]

You can use NaCl to final conc of 0.5M and add EtOH to a final concentration of 70-80%. If you put it in the -20 C freezer for a few hours, it will precipitate easily. There are also methods with isopropanol (rubbing alcohol), which only needs 
35-40%, but salts are less soluble so don't put it in the freezer, just chill to 4 C.  

[jlund256]

DNA precipitation is a fairly forgiving process. 

a) Add 1/10 volume saturated NaCl (5M or stronger), then 2 volumes EtOH.

b) Precipitation works OK at room temperature, but if the amount of DNA is ng, then chilling to -20C helps. 

c) Spin down, wash pellet w/ 70% EtOH to remove salt.

Cheers,

Jim Lund

[Andreas "Mega" Stuermer]

Alright, 

It seems the following protocol worked: 

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. 

[Andreas "Mega" Stuermer]

My centrifuge is not cooled and I did -20°C overnight. I dried it overnight in a beaker covered with aluminium foil 

[Andreas "Mega" Stuermer]

Actually pretty happy with how it turned out. All you need is NaCl (I autoclaved it in a Schott flask), distiled water and 70% & 96% EtOH. 

No fancy salts or liquids required. Although, IIRC in some countries isoprop is easier to get than ethanol