The following is a protocol for purification of DNA that is to be used in the creation of transgenic mice. We wish to thank current and former members of Yale for its availability. Please note that alternate protocols can be found in Manipulating the Mouse Embryo, a laboratory manual, Second Edition, Eds. Hogan, Beddington, Costantini, Lacy Cold Spring Harbor Laboratory Press, 1994.
In purifying the DNA, please use powder free gloves so as to avoid particulate matter that may interfere with DNA microinjection. Also please use MilliQ or an equivalent water source (available upon request).
- Double band plasmid over CsCl/EtBr gradient by equilibrium centrifugation.
- Isolate the construct fragment from the plasmid sequences by appropriate restriction endonuclease treatment followed by horizontal agarose gel electrophoresis, using a Tris-Borate-EDTA buffer system.
- Follow the electroelution protocol as described below.
- Run 20-50ug digested DNA in GTG grade agarose using TBE or TAE buffer.
- Excise band from gel with a clean ethanol wiped razor blade.
- Trim gel strip to fit into the smallest size dialysis membrane (Spectra/pore 4MW cutoff 12-14,000).
- Take a prepared hydrated length of dialysis tubing about 4 cm longer than the length of the gel strip. Close one end with a dialysis bag clip.
- Place 0.5-1.0 ml of 0.5X TBE into the tubing (this will allow the gel strip to slide in smoothly. You can squeeze the liquid up to the top of the tubing while dangling the gel strip to accomplish this).
- Apply the second clip. In so doing you may remove up to 1/2 of the liquid from the tubing, taking care that there are no air bubbles.
- Orient the tubing in a gel box so that it is exactly parallel to the electrodes and perpendicular to the electrical field in the buffer. Cover the tubing with 0.5X TBE. Run the fragment out of the strip and on to the wall of the tubing at 30-50 mA for about 30 min. Monitor progress using a hand-held long wavelength UV illuminator in the darkened room. Stop when the fragment has completely lined up on the inside of the dialysis tubing.
- Reverse the electrodes and run the power supply at the same settings for 0.5-2.0 minutes. Monitor progress again as described above. You should see the EtBr-stained DNA move off of the inside wall of the tubing.
- Remove the top clip and cut off any excess dialysis tubing. Draw off the DNA-containing liquid, trimming the tubing if necessary.
- Add 0.1 volume of 3M NaAc and 2-3 volumes of absolute ethanol. Precipitate for 1 hour or more.
- Resuspend DNA in 40-80 µl injection buffer (10mMTris/0.1mMEDTA, pH 7.4) and pass it through an ion exchange column (e.g., Schleicher & Shuell Elutip columns) according to the manufacturer’s instructions.
- 0.2M NaCl
- 0.02M TRIS pH 7.4
- 0.001M EDTA
- 1.0 M NaCl
- 0.02M TRIS pH 7.4
- 0.001M EDTA
- Pyrogen Free Water
Caution: Never apply negative pressure to the ELUTIP
- Adjust the DNA solution to the "low" buffer conditions using stock solutions of NaCl, Tris and EDTA made with pyrogen free water.
- Cut off the tip of a n Elutip column about 2mm from the white plug in the narrow end. Remove cap from the top of the Elutip.
- Take two 5ml disposable syringes and mark one "low" and one "high". Remove the plunger from the "high" syringe and attach the Elutip using a forceps for leverage. With your pipettor, add 2 ml "High" buffer and replace the plunger. Push the buffer through the Elutip slowly, then force the air trapped in the syringe through the Elutip. DO NOT pull plunger out.
- Remove the Elutip from the "high" syringe and attach it to the plungerless "low" syringe. Put about 5 ml of "low" buffer in the syringe and push this through. The Elutip is now ready for binding the DNA.
- Remove Elutip and then remove the "low" syringe plunger. Attach an Elutip prefilter to the syringe, and then attach the Elutip itself.
- Pipette the DNA/"low" solution into the syringe/prefilter/Elutip assembly.
- Slowly (around 1 ml/min) push the DNA through. Collect the flowthrough and disassemble the syringe from the prefilter. Remove the plunger, replace the syringe body on the prefilter/Elutip, and apply the flowthrough slowly once again.
- Slowly apply 3 ml "low" buffer using the same syringe.
- Push air through the Elutip assembly.
- Attach the "high" syringe body to the Elutip without prefilter.
- Add 0.4 ml "high" buffer to the syringe. Replace the plunger and push the buffer through the Elutip slowly as you collect the eluate.
- Remove the Elutip, disassemble the syringe and replace the Elutip on the syringe body. Repeat the elution with 0.1 ml of "high" buffer.
- Push air through the column to collect any further eluate.
- Ethanol precipitate the pooled eluates by adding 2 volumes of absolute ethanol and centrifuge in a cold-room at 25,000 g for at least one hour.
- After centrifugation aspirate the supernatant ethanol using a flame-drawn pasteur pipette. Dissolve the pellet in 40-80 µl injection buffer (see above) and dialyze.
- Add 5 ml of injection buffer to each of three 60mm plastic petri dishes. Gently float a 0.05um pore size, 13-mm diameter Millipore VMWP 01300 filter in each dish (shiny side up).
- Apply the sample to the first disc and dialyze for 20 minutes. Repeat 2 more times.
- Quantitative the DNA using a series of amounts of digested DNA (e.g., Lambda-HindIII) run on an agarose gel along with 1-2 µl of your dialysate. Adjust the DNA concentration to 5 ng/µl with injection buffer and filter with a 0.2um Millipore SJHV 004NS syringe filter and submit for microinjection.