Team:HKUST/Protocols/gel extraction

Gel extraction

Purpose: To clean up the gel after band cutting from the gel and leave only the desired DNA fragment.

Materials: GEX Buffer, WN Buffer, WS Buffer, Elution Buffer (all provided in FavorPrepTM Gel/PCR DNA Clean-Up Kit)

Procedure:

a.Add 0.5ml GEX buffer into the tube with gel fragment in it.
b.Incubate at 60°C for 5-10mins until the gel is completely dissolved. Invert the tube every 2 mins during the incubation. Stop incubation after the gel is completely dissolved. Let the gel mixture to cool down to room temperature.
c.Place a GP column onto a collection tube. Load no more than 0.7ml gel mixture into each column. Centrifuge for 30s-60s (9000rpm). Discard the flow through.
d.Repeat step c for the excessive gel mixture.
e.Wash the column once with 0.5ml WN buffer by centrifuging for 30s-60s (9000rpm). Discard the flow through.
f.Wash the column once with 0.5ml WS buffer by centrifuging for 30s-60s (9000rpm).
g.Centrifuge for another 3 mins (13200rpm) to remove all the ethanol
h.Place the column onto a new 1.5ml centrifuge tube. Add 15μL -30μL elution buffer onto the center of the membrane. (Preheated the Elution buffer to 60°C).
i.Stand the column for another 3mins and then centrifuge at full speed for 1-2 mins to elute DNA.

Tips:

The gel extraction has a low recovery rate of about 10%. Stand the column longer in step i may give relatively higher recovery rate.

• 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