Hello everyone,

I am looking for some advice on the CTAB method for DNA extraction from faecal samples (from bat droppings). I am following the below written method but I am a bit unclear on the 1% PVP, how much is this to volume? And what are your recommendations on acetone percentage? There are a lot out there, I am looking for an ‘undiluted’ one.

- CTAB buffer (2 % CTAB, 20 mM EDTA·Na2·2H2O, 1.4 M NaCl and 100 mM Tris, pH 8)

- 1% PVP

- Chloroform:isoamyl alcohol 24:1

- AW1 buffer

- Acetone

- EB buffer ( 50 mM Tris-CI (pH 8.1 – 8.2, 15% ethanol (v/v)) (Alternatively TE buffer or EA buffer can be used)

1) Place one guano sample in a microfuge tube and crush with sterile toothpick

2) Incubate samples overnight in 300ul CTAB buffer at 37°C on sample agitator at 400rmp

3) Add 300ul chloroform:isomyl alcohol 24:1 and centrifuge for 10 min at 13rmp to spin down the pellet

4) Transfer the upper aqueous phase only to a clean microfuge tube.

5) Add 1.5 volumes of AW1 to the supernatant

6) Apply the sample to a spin columns and incubate at room temperature for 2 min before spinning

7) Wash columns with 600ul AW1 and centrifuge again

8) Final wash with 300ul acetone

9) Dry columns at room temperature for 5 minutes

10) Elute DNA in 120ul of EB buffer after 2 min of incubation

Thank you very much!

Natasja