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Purify PCR Primers
From Sci-Mate Wiki
Biological and Medical Sciences > Classification by Description > Reagents, Kits and Assays > DNA and RNA > Extraction and Purification| Branch: Protocol-Wiki Type: Protocol Intention: For Publication Read/Write Permissions: Open Access | Authors : 16.09.2010 - John Stewart |
Two methods exist to purify primers following PCR: separation by electrophoresis gel and purification from the gel (by a kit or via DNA silica purification); or PEG precipitation.
This author prefers the use of the gel to separate and then purify, as being more reliable. An alternative would be to run a few ul on a gel (which is normal to check the success of the PCR), then to keep this gel fragment in case the following protocol does not work!
Assuming most molecular biologist know how to run a gel and cut a fragment, here is the protocol for PEG purification adapted from the protocol supplied with the Gateway technology:
- Add 150ul of TE, pH 8 to 50ul solution from PCR.
- Add 100ul of 30% PEG-8000 30mM MgCl2, and vortex thoroughly before centrifuging 10,000 g for at least 15 minutes at room temperature.
- Remove supernatant carefully, the pellet will be invisible.
- Add 50ul of TE in a way that would dislodging the pellet, and allow to resuspend for 1 hour.
Check the concentration on a gel.
