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Protocols GelPurification
From 2008.igem.org
Extraction Procedure
1. Cut out DNA fragment
2. Add 600ul of Buffer QG
3. Incubate at ~50*C until gel has completely dissolved, vortexing occasionally
4. Check mixture is yellow in color
5. Add 100ul of isopropanol and mix by inverting the tube several times
6. Place a purple spin column in a provided 2mL collection tube in a suitable rack and pour mixture in it
800ul maximum per column
7. Centrifuge for 1 min and discard the flow-through and place the spin column in the same collection tube
8. Add 500ul of Buffer QG to the spin column and centrifuge for 1 minute
9. Discard the flow-through. Place the column in a clean 1.5mL microcentrifuge tube
10. Centrifuge for an additional 2 minutes
11. Place the column into another clean 1.5mL microcentrifuge tube
12. To elute DNA, add 50ul of 2mM Tris-Cl for backbone and 30ul 2mM Tris-Cl for insert into the center of the membrane, let stand for 1 minute, and centrifuge for 1 minute
To take away phosphates from backbone...
Add to purified Gel:
5 uL of 10X Antarctic Buffer (1/10 of total volume) 1 uL of Antarctic Phosphatase Enzyme
Incubate this for 15 min @ 37 C
Then heat inactivate for 5 min @ 65 C
Product ready for ligation
