Microinjection DNA Purification

We have found the UltraClean™ GelSpin™ Kit (available from Mo Bio Laboratories, Catalog number 12400-250) is a simple and fast way to obtain microinjection quality DNA. This is not to exclude other methods of DNA purification but to say that we find this is a convenient method that is less onerous than CsCl gradients.

1. Perform restriction digest to liberate transgene from plasmid vector sequences. Final yield should be 10 - 20 micrograms of transgene insert.

2. Separate restriction digest products on agarose gel using either TBE or TAE.

Use either low- or standard- melting temperature agarose.

3. Place gel on transilluminator. Cut out band(s) of interest. Use a clean razor blade or scalpel.

Remove as much excess agarose as possible. Minimize DNA exposure to UV light to prevent photochemical damage (less than 1 minute).

4. Transfer agarose slice(s) to a preweighed tube. Reweigh tube to determine weight of agarose in tube.

5. For each 100 mg of gel, add 300 microliters GelBind buffer.

6. Dissolve at 50 degrees Centigrade for 10 minutes, vigorously vortexing every 2 to 3 minutes. Higher percenteage gels (2%) will take longer to dissolve.

7. Place a spin filter in a 2 ml micro tube and load 700 microliters of the dissolved gel slice onto the cartridge. Spin at maximum speed for 10 seconds in a microcentrifuge. Reload the flowthrough and spin again. Discard the flowthrough after the second spin. The spin filter has a capacity of 20 micrograms DNA, so you can run several 700 microliters loads of dissolved gel slice through a single spin filter.

8. Add 300 microliters of GelWash buffer to the cartridge and spin at maximum speed for 30 seconds in a microcentrifuge. Discard the flowthrough.

9. Replace tube with a fresh microtube. Spin the empty cartridge at maximum speed for 60 seconds in a microcentrifuge to completely remove GelWash buffer .

10. Elute the DNA from the cartridge: Replace tube with a fresh microtube. Add 50 microliters of elution buffer to center of spin filter. Spin at maximum speed for 30 seconds in a microcentrifuge.

Elution buffer: 10 mM Tris-HCl, pH 8.0, 0.02 micron filtered. Check the pH of the elution buffer before just before you use it, best yields are obtained at a pH of 8.0 or greater. The 0.02 micron Anotop syringe filters are available from Whatman.

11. If desired, repeat step 10 to increase yield. We obtain 90% of the DNA in the first elution.

12. Quantitate DNA solution.

13. Verify size and intact condition of DNA on minigel.

14. Store eluted DNA at -20 degrees Centigrade.