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Team:Cambridge/Protocols/DNA Precipitation
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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. | 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. | ||
| - | Resuspend in Elution Buffer. | + | Resuspend in Elution Buffer to give a purified DNA solution. |
===Safety=== | ===Safety=== | ||
| - | + | ||
| + | Ethanol is flammable, care should be taken to perform this experiment away from sources of ignition. | ||
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}} | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}} | ||
Revision as of 09:57, 15 July 2011
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, so will precipitate.
Practice
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).
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.
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.
Resuspend in Elution Buffer to give a purified DNA solution.
Safety
Ethanol is flammable, care should be taken to perform this experiment away from sources of ignition.
