Nov 07, 2017
Version 2
DNA extraction using the ammonium acetate technique V.2
- 1Oviedo University
Protocol Citation: Juan Carlos Illera 2017. DNA extraction using the ammonium acetate technique. protocols.io https://dx.doi.org/10.17504/protocols.io.knycvfw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: November 06, 2017
Last Modified: March 28, 2018
Protocol Integer ID: 8632
Abstract
A simple protocol to extract DNA
Attachments
Before start
If blood is preserved in ethanol, it has to be dried before starting.
Add 50 µl of blood, 200 µl of a DNA extraction solution (which includes tris-HCL 30 mM ph 8, EDTA 10 mM, and 0,4% SDS), and 3 µl proteinase K.
50 µL Blood
200 µL DNA extraction solution
3 µL Proteinase K
Vortex and overnight at 56°.
56 °C
Also incubate 70°C around three hours shaking frequently.
70 °C
03:00:00 Incubation
Add 200 µl AcNH4 4 M. Vortex and incubate 30 minutes. Shake every 10 minutes.
200 µL AcNH4 4 M
00:30:00 Vortex and incubation
00:10:00 Shake
Centrifuge 15 minutes at 13000 rpm.
00:15:00 Centrifugation
Move the supernatant to new tubes.
Add 800 µl of cold EtOH 100%.
800 µL EtOH
Centrifuge 15 minutes at 13000 rpm.
00:15:00 Centrifugation
Remove the supernatant.
Wash the pellet with 800 µl of EtOH 70%.
800 µL EtOH 70%
Centrifuge 5 minutes at 13000 rpm.
00:05:00 Centrifugation
Remove the supernatant and dry.
Add 200 µl TE Buffer and leave 2-3 hours in the oven at 37°C
200 µL TE Buffer
03:00:00 Oven
37 °C