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Team:HKUST/Protocols/gel extraction
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(Difference between revisions)
(New page: 7. 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 (a...) |
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e.Wash the column once with 0.5ml WN buffer by centrifuging for 30s-60s (9000rpm). Discard the flow through. | 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). | 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. | ||
Revision as of 14:00, 26 August 2009
7. 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.
