Protocol for purifying DNA for microinjection
Cesium Chloride: possible mutagen, avoid inhaling and skin contact.
Ethidium Bromide: extremely toxic, avoid inhalation and skin contact
Always wear gloves and lab coat throughout this procedure.
In case of contact, immediately flush eyes or wash skin with copious amounts of water. Seek medical assistance from Boynton Health Service, if necessary (625-7900).
Ethidium Bromide has its own waste and is disposed of in the Shimizu lab. The ethanol waste is disposed of in the appropriate containers found in our lab.
Use a towel to clean up the ethidium bromide and use a UV light to ensure the clean-up was successful.
Procedure:
F Cesium Banding
Add 1 mg plasmid DNA to 7.0 ml of ddH20. Add 0.4 ml Ethidium Bromide (5mg/ml). Weigh this solution. Add X g of Cesium Chloride according to the formula (0.978 x weight in grams) to dissolve. I do this in a 15 ml conical tube. Transfer to a polyallomar tube using a glass pasteur pipette. Fill to the second lip on the tube. Make sure that there is no solution where the cap is going to be placed. If there is some liquid, carefully use a Kimwipe to get rid of it. Push the black cap down and make sure that a nice seal is observed. These steps are critical because the centrifugation could result in a collapsed tube, the sample will be lost and you will have to spend an hour cleaning the rotor. Spin in VTI-75 at 45,000 for 16 hours at 220C. Use the UV light in the darkroom to see the band. Recover the band either using a needle or a pasteur pipette. Minimize light exposure at this step.
Suggestions: Use DNA that is already relatively pure (i.e. from a PEG precipitation).
F Removal of EtBr and Cesium
Extract with an equal volume of water saturated n-butanol (Shimizu lab). The butanol is the top solution. Continue extracting until there is no pink color left and then extract two more times.
Transfer to a corex tube and do two EtOH precipitations. Dilute the sample 1:3 with ddH20, add 5M NaCl to equal 1/20 (if final volume is 4 ml, add 0.2 ml of NaCl) of that volume and add two volumes of ethanol. Freeze (-200C), spin @ 10,000 10 min. Discard supernatant, redissolve pellet in 2 ml H2O, add 0.1 ml 5 M NaCl and 4 ml of EtOH. Freeze, spin @ 10,000 10 minutes. Discard supernatant, rinse with 70% EtOH. Dry until pellet is clear (around 1 hour).
Dissolve in 1 ml of TE, quantitate a 1:40 dilution by Spec. at 260/280.
F Purify Insert
Digest 50 mg of DNA with appropriate enzymes (I use 1 Unit per mg of DNA) in a total volume of 100 ml for one hour or O/N depending on time constraints. Add 20 ml of 6x loading buffer and load into one large well of an 0.8% agarose gel. NO ETBR!!!! I also load cut and a small amount of uncut in the smaller wells to ensure that the digestion worked and to line up the bands more efficiently.
Slice of sides of the well and include the cut/uncut smaller wells with the ladder and stain in EtBr. Align the edges with the gel to assist in excising the band. Purify from agarose using the Qiagen Kit. Depending on how many tubes you need and how much DNA is there will dictate how many beads you need. There can not be more than 0.3 mg of agarose per eppendorf tube. For each tube, do a double elution with H2O and combine all of the tubes together. Spin at maximum speed for 5 seconds to get rid of the beads. Transfer to supernatant to a new tube and repeat this step.
F EtOH precipitation
Add ddH2O to 400 ml, add 20 ml of 5M NaCl add 800 ml EtOH. Freeze, spin @ 14,000 10 min. Rinse with 70% EtOH, dry pellet.
F Dialysis
Resuspend in 1.0 ml of dialysis buffer (5mM Tris, .25 mM EDTA, pH 7.4) Quantitate yield of a neat solution by Spec at 260/280. Recovery depends on the size of the insert. Dilute 1 mg of DNA to 2 mg/ml in dialysis buffer (500 ml), freeze remaining DNA. Transfer to washed, boiled dialysis tubing (Small size, 12-14000 MW cutoff) and dialyze ON x 4L.
F Transfer to clean tubes.
Rinse two clean sterile eppendorf tubes with dd H2O. Transfer dialysate to first clean tube. Spin 1 minute @ maximum speed. Take off top 90% and transfer to the second clean tube. Advise Sandi to spin prior to taking DNA out.
Check concentration and purity of DNA by running 20 ml on a mini-gel with a known quantity of marker.