I am looking for a protocol for PCR Product purification which can remove extra primers, dntps and non specific products as well. Can anyone help?
hello,
PCR products purification solution:
3 M sodium acetate (pH 4.8): 12 ul
40% PEG: 30 ul
200 mM MgCl2: 1.5 ul
total volume mix: 43.5 ul
Steps:
1. add 43.5 ul purification solution into each PCR product tube and vortex.
2. incubate at 4 degree Celciusfor 1 hour to overnight.
3. Centrifuge at 15000 rpm for 15 minutes, room temperature. get rid of supernatant.
4. add 180 ul 80% ethanol, mix, centrifuge same as step 3. get rid of the supernatant
5. dry out the suspension in the clean drawer covered by tissue paper for ca. 2 hours (or you can use dryer vacuum equipment for 15 ins)
6. add 15 ul sterilized MilliQ water, mix by pipette. keep it ta -20 degree celcius.
please go through the material it may help you
*protocol is standardized for Roche High Pure PCR Product Purification Kit
If you know the size of your PCR product, you can use AMPURE XP beads to remove fragments that are smaller and larger than your PCR product. DNA binds to magnetic beads in the presence of PEG. Depending on the concentration of PEG, different sizes of DNA will bind to the beads. It is great for removing smaller fragments such as primer dimers and such. See the link for a detailed protocol. https://www.beckmancoulter.com/wsrportal/techdocs?docname=B37419
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