Isopropanol Precipitation for PCR Purification: Difference between revisions
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To precipitate a PCR product the following method can be used | To precipitate a PCR product the following method can be used | ||
==Procedure== | |||
Take a known volume of PCR product and add one fifth of that known volume as 3M Sodium Acetate (we want a final concentration of 0.3M Sodium Acetate in the solution that will go into the freezer; for 100uL PCR product add 20uL of 3M Sodium Acetate). Next add a volume of isopropanol to the solution that is from 80-100% of the volume of PCR product (continuing the example, you would now add 80uL of isopropanol). | Take a known volume of PCR product and add one fifth of that known volume as 3M Sodium Acetate (we want a final concentration of 0.3M Sodium Acetate in the solution that will go into the freezer; for 100uL PCR product add 20uL of 3M Sodium Acetate). Next add a volume of isopropanol to the solution that is from 80-100% of the volume of PCR product (continuing the example, you would now add 80uL of isopropanol). | ||
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Resuspend in water or you DNA stabilizing buffer of choice. | Resuspend in water or you DNA stabilizing buffer of choice. | ||
==See also== | |||
* [Purification of DNA] - overview page | |||
* [Ethanol precipitation of nucleic acids] | |||
Revision as of 04:18, 25 April 2007
| back to protocols | ||
To precipitate a PCR product the following method can be used
Procedure
Take a known volume of PCR product and add one fifth of that known volume as 3M Sodium Acetate (we want a final concentration of 0.3M Sodium Acetate in the solution that will go into the freezer; for 100uL PCR product add 20uL of 3M Sodium Acetate). Next add a volume of isopropanol to the solution that is from 80-100% of the volume of PCR product (continuing the example, you would now add 80uL of isopropanol).
Place in the -80 degree freezer for at least 15 minutes.
Centrifuge at max speed on a table top centrifuge (typically 14,000-25,000 rpm) for 20 minutes.
CAREFULLY pipette off the supernatant. The PCR product may be difficult to see and quite small depending on the amount of starting material. To remove salts which may interfere with futher manipulations (such as ligation reactions) wash the pellet with 70% ethanol (95% ethanol if the DNA is less than 200 bases in size) and repeat centrifugation step.
Resuspend in water or you DNA stabilizing buffer of choice.
See also
- [Purification of DNA] - overview page
- [Ethanol precipitation of nucleic acids]
