Microinjection DNA Purification

 

Microinjection DNA Purification

We have found the NucleoSpin^TM Extract Kit (available from Clontech , Catalog number K3051-1) 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 buffer NT1. If agarose concentration is greater than 1%, add proportionately more buffer. For example, if a 2% agarose gel is used, add 600 microliters buffer NT1 for each 100 mg of gel.

6. Dissolve at 50 degrees Centigrade for 10 minutes, vigorously vortexing every 2 to 3 minutes.

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

8. Add 750 microliters of buffer NT3 to the cartridge and spin at maximum speed for 60 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 buffer NT3.

10. Elute the DNA from the cartridge: Replace tube with a fresh microtube. Add 50 microliters of preheated (60 degrees Centigrade) elution buffer to cartridge. Spin at maximum speed 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 .

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.