Team:UNICAMP-Brazil/Protocols/Purification of DNA fragments from agarose gels


Topo l2.gif topo_r_igem.gif

Back to Protocols

PureLink™ Quick Gel Extraction Kit - Invitrogen

1. Equilibrate a water bath or heat block to 50°C.

2. Excise the area of the gel containing your desired DNA fragment and minimize the amount of agarose surrounding the DNA fragment.

3. Weigh the gel slice containing the DNA fragment using a scale sensitive to 0.001 g, then place the gel into a polypropylene microcentrifuge tube and add Gel Solubilization Buffer (GS1) as directed below.

4. For ≤2% agarose gels: place up to 400 mg of the excised gel containing the DNA fragment into a 1.5 ml polypropylene tube. Add 30microL Gel Solubilization Buffer (GS1) for every 10microL volume of gel.

5. Place the tube containing the gel slice and GS1 into a 50°C water bath or heat block.

6. Incubate at 50°C for 15 minutes. Invert the tube by hand every 3 minutes to mix and ensure gel dissolution.

7. After the gel slice appears dissolved, incubate for an additional 5 minutes.

8. Place a Quick Gel Extraction Column inside a Wash Tube and load the dissolved gel mixture with DNA onto the center of the Column.

9. Centrifuge at >12,000 × g for 1 minute.

10. Add 500–700 μl Wash Buffer (W1) containing ethanol.

11. Centrifuge at >12,000 × g for 1 minute, then discard the flow-through.

12. Centrifuge again at maximum speed for 2–3 minutes to remove residual Wash Buffer and ethanol. Discard the Wash Tube, and place the Column into a Recovery Tube.

13. Add 50 μl Elution Buffer (E5) to the center of the Column.

14. Incubate for 1 minute at room temperature.

15. Centrifuge at >12,000 × g for 1 minute to elute the purified DNA into the Recovery Tube.

16. Store the purified DNA at -20ºC.