Team:TU Darmstadt/Protocols/Purification of DNA

There are several approches for purifying DNA.

Wizard SV Gel and PCR Clean-Up System

Gel Slice and PCR Product Preparation

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 until gel slice is completely dissolved.

B. Processing PCR Amplifications

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

Binding of DNA

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.

Washing

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.

Elution

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.

For additional protocol information visit the Promega Homepage or click [http://www.promega.com/~/media/Files/Resources/Protocols/Technical%20Bulletins/101/Wizard%20SV%20Gel%20and%20PCR%20Clean-Up%20System%20Protocol.pdf here]