Team:Cambridge/Protocols/DNA Precipitation

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==Rescue Precipitation of DNA==
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==Rescue precipitation of DNA==
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Method for recovering DNA after premature elution whilst following [[Team:Cambridge/Protocols/Gel Extraction of DNA |Gel Extraction of DNA]].  
Method for recovering DNA after premature elution whilst following [[Team:Cambridge/Protocols/Gel Extraction of DNA |Gel Extraction of DNA]].  
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===Practice===
===Practice===
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If elution buffer is added before washing with buffer PE, elute the DNA solution into a clean tube.  Add 112.5 µl of sodium acetate to elutant.  Total volume in tube is ~850 µl, so spilt into 3 microcentrifuge tubes to allow addition of more liquid (approx 200 µl in each).
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:# If elution buffer is added before washing with buffer PE, elute the DNA solution into a clean tube.   
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:# Add 112.5 µl of sodium acetate to elutant.   
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Add 3x volume of ethanol (in this case, 600 µl to each tube).  Vortex to mix, then centrifuge for 20 minutes at 13 000 RPM.   
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:#* '''Tip: Total volume in tube is ~850 µl, so spilt into 3 microcentrifuge tubes to allow addition of more liquid (approx 200 µl in each).'''
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:# Add 3x volume of ethanol (in this case, 600 µl to each tube).  Vortex to mix, then centrifuge for 20 minutes at 13,000 RPM.   
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Pipette off liquid, taking care not to disturb pellet. Attempt to remove as much liquid as possible, as ethanol will prevent resuspension of the DNA. Leave tubes on side to allow ethanol to evaporate, taking care that the pellet does not completely dry out.
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:# Pipette off liquid, taking care not to disturb pellet.  
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:#* '''Tip: Try to remove as much liquid as possible, as ethanol will prevent resuspension of the DNA.'''
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Resuspend in Elution Buffer to give a purified DNA solution.
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:# Leave tubes on side to allow ethanol to evaporate, taking care that the pellet does not completely dry out.
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:# Resuspend in Elution Buffer to give a purified DNA solution.
===Safety===
===Safety===
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Ethanol is flammable, care should be taken to perform this experiment away from sources of ignition.   
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Ethanol is flammable, so care should be taken to perform this experiment away from any possible sources of ignition.   
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Latest revision as of 20:21, 21 September 2011

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Contents

Rescue Precipitation of DNA

Method for recovering DNA after premature elution whilst following Gel Extraction of DNA.

Theory

Addition of positive sodium ions mitigates repulsive interactions of the negatively charged DNA backbone, allowing DNA to coelesce. In the presence of positive ions, DNA is insoluble in alcohols and so will precipitate out.

Practice

  1. If elution buffer is added before washing with buffer PE, elute the DNA solution into a clean tube.
  2. Add 112.5 µl of sodium acetate to elutant.
    • Tip: Total volume in tube is ~850 µl, so spilt into 3 microcentrifuge tubes to allow addition of more liquid (approx 200 µl in each).
  3. Add 3x volume of ethanol (in this case, 600 µl to each tube). Vortex to mix, then centrifuge for 20 minutes at 13,000 RPM.
  4. Pipette off liquid, taking care not to disturb pellet.
    • Tip: Try to remove as much liquid as possible, as ethanol will prevent resuspension of the DNA.
  5. Leave tubes on side to allow ethanol to evaporate, taking care that the pellet does not completely dry out.
  6. Resuspend in Elution Buffer to give a purified DNA solution.

Safety

Ethanol is flammable, so care should be taken to perform this experiment away from any possible sources of ignition.

Back to Protocols

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