By Kenzie Chilcott and Angus Ball

Introduction

Once a DNA extraction is complete an Ethanol precipitation (EtOH PPT) is a common technique to purify and concentrate DNA

EtOH PPT is used to eliminate the solvation shell surrounding the nucleic acid, such as phenols and guanidium chloride.

Currently when quantifying extracted DNA there has been a common trend of a low 260/230 ratio when using NORGEN soil DNA extraction kits. That ratio indicates the presence of unwanted organic compounds that may interfere with down-stream processes or proper quantification. Once EtOH PPT is complete the 260/230 ratio should be near 2. The 260/280 ratio is reflective of purity of biomolecule. 1.8 is considered pure for DNA, 2 is considered pure for RNA. Values lower than this suggest protein contamination. See Assessment of Nucleic Acid Purity for more information

What you will need:

• Dead air box (everything will be done inside the dead air box)

• Sodium Acetate (3M)

• 95 % Ethanol at -20˚C

• 70 % Ethanol at 4˚C

• Milli Q o All solution should be stored in clean jars free of DNase and RNase.

  Preparation Instructions

1. Complete all steps in Dead Air box following Dead Air Box

2. Thaw out previously extracted DNA (eg. 50 ul). Mix with vortex then spin down with centrifuge

3. Add 1/10th of initial volume of 3M Sodium Acetate (eg. 5 ul)

4. Add 2.5 of total volume of -20˚C of 95 % ethanol (eg. 135.5 ul)

5. Mix samples with vortex and spin down using a centrifuge

6. Incubate samples overnight at -20 ˚C

   Precipitation

7. Centrifuge samples at max speed (21380 rcf) at 4˚C for 30 minutes

8. Removed supernatant

9. Add 0.5 mL of 4˚C 70 % Ethanol

10. Briefly mix with vortex

11. Centrifuge samples at max speed (21380 rcf) at 4˚C for 30 minutes

12. Remove supernatant wihtout disturbing DNA pellet (You won't be able to see the pellet)

13. Repeat steps 3-6

14. Spin down samples

15. Air Dry for 20-30 minutes

16. Resuspend DNA into Milli Q (eg. 20 ul)