The 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 B) Low Melting Point Agarose Purification 1) Make 2% low melting point (LMP) agarose gel in TAE + Ethidium bromide. Allow gel to solidify at 5oC and store at 5oC until use. 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. 4) Ethanol Precipitation of Nucleic Acids from Gelase 5) Extract purified DNA with Phenol/Chloroform (Maniatis E.3) 6) Final Ethanol Precipitation 7) Cleanup gel-purified DNA by one of the following methods (detailed below): * CsCl centrifugation Cleanup of Gel-Purified DNA by S&S Elutip reg. Method 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 -70oC for 30 min. Pellet DNA in microfuge (12,000 x g for 30 min) and discard supernatant. Rinse pellet with cold (-20oC) 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 Centrifugation 1) 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. |
→如果您认为本词条还有待完善,请 编辑词条
词条内容仅供参考,如果您需要解决具体问题
(尤其在法律、医学等领域),建议您咨询相关领域专业人士。
0