Team:TU Darmstadt/Protocols/Purification of DNA
From 2012.igem.org
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
- Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5ml microcentrifuge tube.
- 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
- Add an equal volume of Membrane Binding Solution to the PCR amplification.
Binding of DNA
- Insert SV Minicolumn into Collection Tube.
- Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
- 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]