Yale University

Preparation of targeting constructs for electroporation

Targeting construct plasmids must be linearized prior to submission for electroporation. Standard methods of plasmid purification, such as CsCl gradient banding or commercial plasmid prep kits, are acceptable. Purified plasmid should be digested to completion with an appropriate restriction enzyme, extracted with phenol:chloroform (or phenol followed by chloroform, if preferred), ethanol precipitated, and resuspended at 1 µg/µl in TE or H₂O. 25 µg of DNA is used per electroporation; we request that you submit 50 µg, in case re-electroporation is necessary.

Preparation of DNA from ES cells in 24-well plates

Many protocols are suitable for isolating DNA from ES cells of sufficient quality for PCR and a Southern blot. This is a straightforward, reliable protocol (from Manipulating the Mouse Embryo, Hogan et. al. 1994) which we can recommend, but investigators should use any protocol they feel comfortable with. Commercial genomic DNA prep kits have also been used with satisfactory results. Feedback about alternate protocols that may be easier or cheaper is welcomed.

From plates containing confluent ES cells:

• aspirate medium, wash once with PBS, aspirate
• add 200 µl lysis buffer; seal plate with parafilm, incubate at 55° a few hours to overnight
• transfer lysate to microfuge tubes; add 100 µl saturated NaCl and shake vigorously
• spin at 3000g for 15 minutes; transfer supernatant to new tube
• add 2 volumes of 100% ethanol at room temperature; invert several times (precipitate should be visible)
• spool out DNA (pipette tip, or heat-sealed end of pasteur pipette); rinse attached pellet in 70% ethanol, dry a few seconds, resuspend in 50 µl TE

Lysis Buffer:	150 mM NaCl 2 mM EDTA (pH 8) 1% SDS 20 mM TRIS-HCl (pH 8) 50 μg/ml proteinase K

Saturated NaCl:	add solid NaCl (with stirring) to 5M NaCl until it no longer dissolves.

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Purification of transgenic constructs for microinjection

The protocols below have proven effective at providing high quality DNA for pronuclear microinjection. In addition, the TMS also recommends the use of QIAGEN QiaQuick and QiaEX II kits for cleanup of electrophoretically separated transgenic cassettes.

Method 1 | Method 2

Method 1

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).

1. Double band plasmid over CsCl/EtBr gradient by equilibrium centrifugation.
2. 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.
3. Follow the electroelution protocol as described below.

Electroelution protocol

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 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.
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 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 (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.

DNA binding

1. Remove Elutip and then remove the "low" syringe plunger. Attach an Elutip prefilter to the syringe, and then attach the Elutip itself.
2. Pipette the DNA/"low" solution into the syringe/prefilter/Elutip assembly.
3. 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.
4. Slowly apply 3 ml "low" buffer using the same syringe.
5. Push air through the Elutip assembly.

DNA elution

1. Attach the "high" syringe body to the Elutip without prefilter.
2. 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.
3. Remove the Elutip, disassemble the syringe and replace the Elutip on the syringe body. Repeat the elution with 0.1 ml of "high" buffer.
4. Push air through the column to collect any further eluate.
5. 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.
6. 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.

Dialysis

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 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.

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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 65-70°C 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 100 µl 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/µl 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:

Method 1 | Method 2