Team:UNICAMP-Brazil/Protocols/Purification of DNA fragments from agarose gels
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- | PureLink™ Quick Gel Extraction Kit - Invitrogen | + | {{:Team:UNICAMP-Brazil/inc_topo}} |
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+ | [[Team:UNICAMP-Brazil/Protocols|Back to Protocols]] | ||
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+ | ==PureLink™ Quick Gel Extraction Kit - Invitrogen== | ||
1. Equilibrate a water bath or heat block to 50°C. | 1. Equilibrate a water bath or heat block to 50°C. | ||
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2. Excise the area of the gel containing your desired DNA fragment and minimize the amount of agarose surrounding the DNA fragment. | 2. Excise the area of the gel containing your desired DNA fragment and minimize the amount of agarose surrounding the DNA fragment. | ||
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3. Weigh the gel slice containing the DNA fragment using a scale sensitive to 0.001 g, then place the gel into a | 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. | polypropylene microcentrifuge tube and add Gel Solubilization Buffer (GS1) as directed below. | ||
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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. | 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. | ||
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5. Place the tube containing the gel slice and GS1 into a 50°C water bath or heat block. | 5. Place the tube containing the gel slice and GS1 into a 50°C water bath or heat block. | ||
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6. Incubate at 50°C for 15 minutes. Invert the tube by hand every 3 minutes to mix and ensure gel dissolution. | 6. Incubate at 50°C for 15 minutes. Invert the tube by hand every 3 minutes to mix and ensure gel dissolution. | ||
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7. After the gel slice appears dissolved, incubate for an additional 5 minutes. | 7. After the gel slice appears dissolved, incubate for an additional 5 minutes. | ||
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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. | 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. | ||
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9. Centrifuge at >12,000 × g for 1 minute. | 9. Centrifuge at >12,000 × g for 1 minute. | ||
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10. Add 500–700 μl Wash Buffer (W1) containing ethanol. | 10. Add 500–700 μl Wash Buffer (W1) containing ethanol. | ||
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11. Centrifuge at >12,000 × g for 1 minute, then discard the flow-through. | 11. Centrifuge at >12,000 × g for 1 minute, then discard the flow-through. | ||
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12. Centrifuge again at maximum speed for 2–3 minutes to remove residual Wash Buffer and ethanol. Discard the | 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. | Wash Tube, and place the Column into a Recovery Tube. | ||
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13. Add 50 μl Elution Buffer (E5) to the center of the Column. | 13. Add 50 μl Elution Buffer (E5) to the center of the Column. | ||
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14. Incubate for 1 minute at room temperature. | 14. Incubate for 1 minute at room temperature. | ||
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15. Centrifuge at >12,000 × g for 1 minute to elute the purified DNA into the Recovery Tube. | 15. Centrifuge at >12,000 × g for 1 minute to elute the purified DNA into the Recovery Tube. | ||
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16. Store the purified DNA at -20ºC. | 16. Store the purified DNA at -20ºC. | ||
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+ | {{:Team:UNICAMP-Brazil/inc_rodape}} |
Latest revision as of 00:53, 17 August 2009
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