Team:LMU-Munich/Notebook/Protocols/12 Gel extraction or PCR Clean up

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<b>Gel extraction or PCR Clean Up</b>

Source:http://www.biodee.net/UploadFile/Up-2009-11-26_633948249752287808-a9281.pdf

Note: from 10ng to 40µg of DNA can be bound by the membrane (don't try to eluate more, as membrane could be blocked)


<b>Gel Slice and PCR Product Preparation</b>

A. Dissolving the Gel Slice

1. Following electrophoresis, excise DNA band from gel and place gel slice in a
1.5ml microcentrifuge tube.

2. Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and
incubate at 50–65°C (we used 95°C as the agar didn't melt at 60°C) until gel slice is completely dissolved.


B. Processing PCR Amplifications

1. Add an equal volume of Membrane Binding Solution to the PCR amplification.


<b>Binding of DNA</b>

1. Insert SV Minicolumn into Collection Tube.

2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn
assembly. Incubate at room temperature for 1 minute.

3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert
Minicolumn into Collection Tube.


<b>Washing</b>

4. Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g
for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection
Tube.

5. Repeat Step 4 with 500μl Membrane Wash Solution. Centrifuge at 16,000 × g
for 5 minutes.

6. Empty the Collection Tube and recentrifuge the column assembly for 1 minute
with the microcentrifuge lid open (or off) to allow evaporation of any residual
ethanol.


<b>Elution</b>

7. Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube.

8. Add 50μl of Nuclease-Free Water to the Minicolumn. Incubate at room
temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute.

9. Discard Minicolumn and store DNA at 4°C or –20°C.

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