Team:HKUST/Protocols/PCR cleanup

PCR product clean-up

Purpose: To clean up primers, dNTPs, enzymes, short-failed PCR products in the PCR reaction.

Materials: FAPC Buffer，Wash Buffer, Elution Buffer, PCR Product (all provided in FavorPrepTM PCR Clean-Up Kit)

Procedure:

a.Transfer 30μL of PCR product and add 5 volumes of FAPC buffer to a 1.5mL microcentrifuge tube, then mix well by vortexing.
b.Place a FAPC Column into a Collection Tube and transfer the sample mixture to FAPC Column.
c.Centrifuge at 6,000 rpm for1 min then discard the flow-through.
d.Add 750 μl of Wash Buffer (ethanol added) to FAPC Column. Centrifuge at 6,000 rpm for 1 min, then discard the flow-through.
e.Centrifuge at 14,000 rpm for an additional 3 min to dry the column.
f.Place FAPC Column into an Elution Tube.
g.Add 40 μl of Elution Buffer or ddH2O (pH 7.0~8.5) to the membrane center of FAPC Column. Stand FAPC Column for 2 minutes.
h.Centrifuge at 14,000 rpm for 1 min to elute the DNA.

Tips:

In step f, for effective elution, make sure that the elution solution is dispensed onto the membrane center and is absorbed completely.

• Agarose gel preparation and gel electrophoresis
• PCR (Taq, Vent)
• PCR product clean-up
• Enzyme digestion (vector, insert)
• Plasmid DNA extraction
• Gel cutting
• Gel extraction
• Ethanol precipitation
• Ligation
• Transformation to E.coli
• Transformation to yeast
• Cell lysis
• Yeast genomic DNA extraction
• Western blotting
• GFP testing