DNA ultrapurificationThe success of DNA purification can have profound consequences on the success or failure of any injection effort. This summary of techniques from several different sources was compiled by Brad Preston at the University of Utah.

PURIFICATION OF DNA FOR MICROINJECTION

(Methods in Enzymology p. 775; Manipulating the Mouse Embryo p. 159)

Isolation of Coding Elements by Agarose Gel Electrophoresis

1) Restrict your clone using restriction sites that maximize separation of the coding elements (transgene + desired 5' and 3' sequences) from vector sequences (Note: some vector sequences appear lethal to embryos). 2) Electrophorese sample of restricted material on 0.8% agarose to check cutting efficiency along with an appropriate marker and an uncut sample of DNA in question. 3) Purify restricted sample by one of the two methods (A or B) below: A) Qiagen-gel purification kit

1) Electrophorese restricted DNA on 0.8% agarose 2) cut out appropriate bands 3) Follow Qiagen kit instructions

B) Low Melting Point Agarose Purification

1) Make 2% low melting point (LMP) agarose gel in TAE + Ethidium bromide. Allow gel to solidify at 5o C and store at 5o C until use. 2) Electrophorese slowly at 5o C (slow and cold to avoid melting of LMP). 3) Extract DNA from Gelase ("Fast Protocol" according to Gelase manufacturer)

a) Cut out the desired band of the LMP-agarose and trim excess agarose--Use long wavelength UV lamp to minimize damage to ethidium bromide-stained nucleic acids. b) Weigh the gel slice in a tared tube c) Add 1 ul 50x Gelase buffer per 50 mg of gel (1 ul of molten agarose=1mg of gel) d) Melt the gel slice completely at 70o C e)Equilibrate the molten agarose at 45o C for 20 minutes. f) Add the appropriate amount of Gelase (1 unit/300 mg of 1% LMP-agarose in TAE) and incubate at 5o C for at least one hour--may incubate overnight.

4) Ethanol Precipitation of Nucleic Acids from Gelase

a) Add one volume of 5M ammonium acetate. b) Add 4 volumes of room temperature 100% ETOH; gently mix c) Centrifuge at 12,000g at room temperature for 30 minutes. DO NOT USE A CENTRIFUGE THAT IS COLD. d) Remove supernatant. e) Add 3 ml 70% ETOH, gently swirl and centrifuge at 12,000g for 30 minutes f) Remove ETOH and dry the pellet g) Resuspend in 1 ml TE buffer

5) Extract purified DNA with Phenol/Chloroform (Maniatis E.3)

a) Add one volume phenol (pre-equilibrated with Tris buffer, pH 8.0) b) Centrifuge 10 minutes @10,000 RPM, room temperature. c) Carefully remove aqueous layer and add equal volume of CHCl3 :Isoamyl Alcohol (24:1). d) Centrifuge 10 minutes @10,000 RPM, room temperature. e) Remove aqueous phase and extract with water-saturated diethyl ether (Maniatis E.4).

6) Final Ethanol Precipitation

a) Add 1/10 volume 3M NaAcetate and 2 volumes of ice-cold 100% EtOH; gently mix and incubate at -70o C for 30 minutes. b) Centrifuge at 10,000 RPM for 30 minutes at 4o C c) Discard supernatant d) Wash pellet with one volume ice-cold 70% EtOH e) Centrifuge at 10,000 RPM for 5 minutes at 4o C f) Discard supernatant and air dry pellet g) Resuspend in 2.4 ml TE buffer pH 8.0

7) Cleanup gel-purified DNA by one of the following methods (detailed below):

* CsCl centrifugation * S&S Elutipreg. minicolumns

Cleanup of Gel-Purified DNA by S&S Elutip reg. Method Low Salt Buffer High Salt Buffer 0.2 M NaCl 1.0 M NaCl 20 mM Tris*HCl 20 mM Tris*HCl 1.0 mM EDTA 1.0 mM EDTA pH 7.4 pH 7.4 20 ml 5 M NaCl 100 ml 5 M NaCl 10 ml 1 M Tris*HCl 10 ml 1 M Tris*HCl 1 ml 500 mM EDTA 1 ml 500 mM EDTA 400 ml dH20 300 ml dH20 pH to 7.4 pH to 7.4 Make up to 500 ml Make up to 500 ml Autoclave Autoclave

Procedure

I. Preparation of Minicolumn

Remove tip protector on the Elutip-d and cut off the tip approximately half-way between the tip and frit supporting the column matrix. Remove the protector cap on the top of the minicolumn. (Consider equilibration with low salt buffer for 2 hours to increase recovery.) Load a Luer-lock syringe with 5 ml of low salt buffer and attach to minicolumn. Wash matrix by pushing buffer through matrix at approximately 0.5-1.0 ml/min.II. Binding of DNA to Minicolumn Reload syringe with DNA sample which has been resuspended in low salt buffer (5, 10 or 20 ml). Attach the Elutip pre-filter to the column. Attach syringe to pre-filter and column and slowly pass all of the sample through the filter and minicolumn. Slow flow of 1-2 drops per second is necessary for efficient binding of the DNA to column.III. Washing the Column Reload the syringe with 2-3 ml low salt buffer. Reconnect syringe to column and push low salt buffer through. Remove the syringe and discard the pre-filter.IV. Elution of DNA from Column Load the syringe with 0.4 ml high salt buffer and reattach to the column. Elute the DNA from the minicolumn by passing the high salt buffer through the column into a 1.5 ml microfuge tube. A second elution is optional. Precipitate DNA with 2 volumes ice-cold EtOH at -70o C for 30 min. Pellet DNA in microfuge (12,000 x g for 30 min) and discard supernatant. Rinse pellet with cold (-20o C) 70% EtOH and respin at 12,000 x g for 30 min. Discard supernatant, dry off EtOH residue, and resuspend pellet in filtered (0.22 u) injection buffer. Quantify DNA by OD260 and dilute in filtered (0.22 u) injection buffer to a final concentration of 1-2 ng DNA/ul.IMPORTANT NOTE!! THE INJECTION BUFFER MUST BE FREE OF PARTICULATE MATTER AND THUS MUST BE FILTERED THROUGH A 0.22uM FILTER PRIOR TO USE. Cleanup of Gel-Purified DNA by CsCl Centrifugation1) Add exactly 3 gms of CsCl to the 2.4-ml solution of your DNA. IMPORTANT NOTE!! You are highly recommended to use Suprapure reg. CsCl from Merck (catalog # 2039) or equivalent. Impurities are lethal to single-cell mouse embryos.2) Mix thoroughly and transfer to 13x51mm polyallomer ultracentrifuge tubes for SW50.1 rotor3) Top tube off with mineral oil (approximately 2 ml)4) Centrifuge at 20o C / 45 K / 40 hours in SW50.1 rotor5) Collect small fractions of approximately 15 drops / fraction6) Determine fraction(s) containing DNA of interest by electrophoresis on 0.8% agarose gel being sure to include an appropriate marker.7) Pool desired fractions and dialyze 3 times against 4 liters of injection buffer (10 mM NaCl, 5mM Tris pH 7.4, 0.1mM EDTA).8) Determine concentration of DNA via optical density. (Note: DNA concentration can also be estimated by ethidium-staining intensity after agarose gel electrophoresis along with DNA standards of identical length and known concentration.)9) Dilute in filtered injection buffer to obtain a final concentration of 1-2 ng DNA/ul.