Isopropanol Precipitation for PCR Purification: Difference between revisions
No edit summary |
No edit summary |
||
| Line 1: | Line 1: | ||
To precipitate a PCR product the following method can be used | To precipitate a PCR product the following method can be used | ||
Take a known volume of PCR product and add one fifth of that known volume as 3M Sodium Acetate (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). | ||
Place in the -80 degree freezer for at least 15 minutes. | Place in the -80 degree freezer for at least 15 minutes. | ||
Revision as of 11:56, 8 December 2006
To precipitate a PCR product the following method can be used
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 will be difficult to see and quite small. Resuspend in water or you DNA stabilizing buffer of choice.
