DNA PURIFICATION

Method 1:

1. Run 20-50ug digested DNA in GTG grade agarose using TBE or TAE buffer.
2. Excise band from gel with a clean ethanol wiped razor blade.
3. Trim gel strip to fit into the smallest size dialysis membrane (Spectra/pore 4MW cutoff 12-14,000).
4. 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.
5. 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).
6. 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.
7. 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 ½ hour. 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.
8. 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.
9. Remove the top clip and cut off any excess dialysis tubing. Draw off the DNA-containing liquid, trimming the tubing if necessary.
10. Add 0.1 volume of 3M NaAc and 2-3 volumes of absolute ethanol. Precipitate for 1 hour or more.
11. Resuspend DNA in 40-80 ul 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 (see below).

Caution: Never apply negative pressure to the ELUTIP

1. Adjust the DNA solution to the "low" buffer conditions using stock solutions of NaCl, Tris and EDTA made with pyrogen free water.
2. 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.
3. 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.
4. 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.

1. 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).
2. Apply the sample to the first disc and dialyze for 20 minutes. Repeat 2 more times.
3. Quantitative the DNA using a series of amounts of digested DNA (e.g., Lambda-HindIII) run on an agarose gel along with 1-2 ul of your dialysate. Adjust the DNA concentration to 5 ng/ul with injection buffer and filter with a 0.2um Millipore SJHV 004NS syringe filter and submit for microinjection.

Method 2
(Kindly provided by one of our users)

Phenol Extraction of DNA Fragment from Low Melting Point Agarose (all at room temp)

1. Cut 25ug of DNA using appropriate restriction enzymes and separate insert and vector by electrophoresis in low melting point agarose (FMC brand)
2. Cut out band using razor blade taking care to minimize gel volume and weigh. Add equal volume of 0.3M NaCl/TE and heat at 65-70oC for 30 min.
3. Extract with one volume of Tris-buffered Phenol. Spin for 10 min in microfuge and take aqueous phase. Back extract with 1/2 volume of 0.3M NaCl/TE.
4. Pool aqueous phases and extract with phenol/chloroform (Repeat this step if aqueous phase is not clear after this step).

Purification on PrePac Column (BRL)
(Allow all solutions to flow through column by gravity flow)

Precipitate DNA by adding 2 volumes of EtOH. Wash pellet once with ice-cold 70% EtOH. Dry pellet in speed-vac. Resuspend in 100ul of DNA Injection Buffer.

N.B. For large fragments the following is recommended:

Dot Dialysis

1. Add several ml of DNA Injection Buffer to a disposable petri dish.
2. Float a 0.1um Millipore filter on buffer and leave for 5 min to equilibrate.
3. Gently pipette DNA solution onto filter and leave for 1.5-2 hrs. Remove DNA solution and attempt to recover as much volume as possible. Add half the volume of solution recovered to filter, leave for several min and recover this sample also(improves yield).
4. Spin DNA solution for 30 min in microfuge and take ONLY TOP 90% of solution (this step is very important to remove particulates that will block injection pipette, failure to adequately remove particulates may result in having your DNA sample returned to you for re-purification).
5. Dilute an aliquot of DNA solution to close to the final concentration of approx. 5 ng/ul and estimate concentration exactly by comparison with known standards on an agarose gel.

Solutions for Method 2:
(Sterilize by filtration)

DNA Injection Buffer:

1M NaCl/TE:

0.2M NaCl/TE:

This information adapted for the WEB and maintained by Greg Blasko
Last modified: July 12, 2000