Microinjection DNA Purification

We have found the NucleoSpin® Extract II  Kit (available from Macherey-Nagel, Catalog number 740609.50) 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 time consuming 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 200 microliters buffer NTI. If agarose concentration is greater than 1%, add proportionately more buffer. For example, if a 2% agarose gel is used, add 400 microliters buffer NTI for each 100 mg of gel.

6. Dissolve at 50 degrees Centigrade for 10 minutes, vigorously vortexing every 2 to 3 minutes until the agarose is completely dissolved.

7. Place a NucleoSpin cartridge in a 2 ml micro tube and load 750 microliters dissolved gel slice onto the cartridge. Spin at 11,000xg  for 60 seconds in a microcentrifuge. Discard the flowthrough. The cartridge has a capacity of 15 micrograms DNA, so you can run several 750 microliters loads of dissolved gel slice through a single cartridge.

8. Filter 750 microliters of buffer NT3 through Anotop 10 0.02 micron filters (GE Healthcare Life Sciences, Catalog number 6809-1002). Add buffer to the cartridge and spin at 11,000xg  for 60 seconds in a microcentrifuge. Discard the flow-through.

9. Replace tube with a fresh micro-tube. Spin the empty cartridge at 11,000xg for 60 seconds in a microcentrifuge to completely remove buffer NT3.

10. Elute the DNA from the cartridge: Replace tube with a fresh micro-tube. Add 50 microliters of preheated (60 degrees Centigrade) elution buffer to cartridge and incubate one minute. Spin at 11,000xg  for 60 seconds in a microcentrifuge.

Elution buffer: 10 mM Tris-HCl, pH 8.5, 0.02 micron filtered. Check the pH of the elution before just before you use it, best yields are obtained at a pH of 8.5 or greater. The 0.02 micron Anotop syringe filters are available from Whatman (GE Healthcare Life Sciences).

If you have problems with particulates plugging the micoinjection needles, pre-filter the wash and elution buffers with the 0.02 uM filters. Do not filter the DNA through the filters, the small pore size will trap DNA molecules.

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.

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