Team:Newcastle/Project/Labwork/OurProtocols/EthanolPrecip

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<font color="blue">''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.''</font>
<font color="blue">''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.''</font>
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* Place the tubes of DNA sample solutions into the ice bucket and leave them for 15 minutes.
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<font color="blue">''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.''</font>
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* 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.
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<font color="blue">''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''</font>.
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Revision as of 19:55, 9 August 2009

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 or buffer (10mM)
  • Ice bucket (with ice)
  • Eppendorf Tubes
  • Centrifuge


Procedure

  • 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.

  • 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.

  • 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.

  • 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.



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