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Ethanol Precipitation

This protocol is designed to remove the ethanol which is likely to be present in a DNA sample once it has been midi-prepped (the ethanol is found in Wash Solution 2).

What you will need

• 3M Sodium Acetate (pH 5.2)
• 100% ethanol
• 70% ethanol
• Deionised water
• Ice bucket (with ice)
• Eppendorf Tubes
• Centrifuge

Procedure

1) Measure the DNA sample volume and then calculate 1/10 of this volume. This is the amount of sodium acetate that needs to be added to the sample - pipette this volume of sodium acetate into the DNA sample.
For example if the DNA sample measures 2ml calculate 10% of this volume (which is 0.2ml). Take up 0.2ml of the sodium acetate and add it to the DNA sample - this should make up a total volume of 2.2ml.

2) Add 100% ethanol to the solution. To work out the amount of 100% ethanol needed, multiply the volume of the DNA sample (plus sodium acetate) by 3.
For example if the DNA sample (plus sodium acetate) makes up a volume of 2.2ml, multiply the 2.2ml by 3, which gives the amount of 6.6ml. Add this volume of ethanol to the DNA (plus sodium acetate) solution, which should now give a total volume of 8.8ml.

3) Place the tubes of DNA sample solutions into the ice bucket and leave them for 15 minutes.
Alternatively, if you are pushed for time, you could incubate the samples in the fridge overnight and carry out the rest of this protocol the next day.

4) Split the samples into Eppendorf tubes - each tube should contain 1ml of the sample. The next stage in this protocol involves centrifugation, therefore it is vital that the DNA sample solutions are transferred to Eppendorf tubes.
For example if we have a total volume of 8.8ml DNA sample solution, we will have to split the solution into 8 Eppendorf tubes.

5) Transfer the Eppendorf tubes to the centrifuge and spin the samples for 20 mins (at the maximum speed of 13,000 rpm)
To make things easy for the next step, when the tubes are placed in the centrifuge make sure that the tabs of the lids are facing the outside. By doing it this way you can be assured that the DNA pellet will have deposited on the opposite side in the Eppendorf tubes. In cases where the pellet isn't visible this is a really helpful consideration.

6) Remove the supernatant making sure that the pellet does not disappear with it.

7) Add 70% ethanol to the DNA pellet without disturbing it and then discard the ethanol, making sure that the pellet is still present in the tube.
Alternatively after placing the 70% ethanol (which serves as a rinse) into the tubes, you could centrifuge the DNA and 70% ethanol for about 15-20 minutes. This is to ensure that the pellet is secured onto the sides of the Eppendorf tubes and not washed off by the ethanol.

8) Ensure that the pellet is completely dried - remove the droplets of ethanol from each tube using a pipette tip, use a vacuum pump to remove any remaining ethanol in the tubes and then leave the tubes on the bench (preferably on paper towels) for a few minutes to dry.

9) Resuspend the pellets in 15 microlitres of sterile deionised water.

10) Either use the DNA now or store it in the freezer.