Protocols GelPurification

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

Protocols