Team:UIUC-Illinois/Notebook/Protocols/GelPurification

Select Protocol

• Bootcamp Protocols
• Subculturing Plates
• Making Electrocompetent E.Coli
• Making TAE Buffers
• Making Electrophoresis Gels
• Gel Purification
• Inoculation
• Miniprep
• PCR Protocols
• Digestions
• Ligations
• Transformation of E.Coli
• Storage of Cells

Gel Purification

What you need:

- The sample of interest, typically a DNA double-digest (50 uL)
- A low-melt gel
- Razor blade
- Autoclaved 1.5 mL centrifuge tubes
- Agarose-dissolving buffer (ADB)
- Big Zymo-spin columns in collection tubes
- DNA wash buffer
- Autoclaved MilliQ water

Procedure:

1. Place each sample into a 1.5 mL centrifuge tube
2. To each tube, add 3 volumes of ADB (i.e for 100 uL gel slice add 300 uL ADB)
3. Incubate at 37-55˚C in the warm water bath until the gel is completely dissolved (if DNA fragment is larger than 8kb, add one additional gel volume of water to the tube following incubation for better DNA recovery)
4. Transfer the melted agarose solution to a Zymo-spin column in a collection tube
5. Centrifuge for 30 seconds and discard liquid flow-through
6. To the column, add 200uL wash buffer
7. Centrifuge for 30 seconds and discard the liquid flow-through
8. Repeat steps 6-7
9. Centrifuge the empty column for 30 seconds
10. Place column in a new 1.5 mL centrifuge tube
11. Gently add 30uL autoclaved water to the column
12. Let column stand for 2 minutes
13. Centrifuge for 30-60 seconds to elute pure DNA into the tube and store at -20˚C until use