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        DNA转化实验指导

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        1372

         

         

         

         

         

        DNA转化实验指导

        CONTENT
         
      • Transformation-Competent E. coli preparation

         

         

        • Rubidium chloride method

         

        • Inoue "ultra-competent" method

         

         

      • Cosmid packaging protocol

         

         

         

      • DNA Ligation and Transformation Protocols

        [NextPage]

        Rubidium Chloride method for Transformation Competent E. coli

        Procedure

        1. Inoculate 1 ml from overnight culture into 100 ml Psi broth (scale up or down as needed). Incubate at 37 C with aeration to A550=0.48

        2. Ice 15 min.

        3. Pellet cells in appropriate centrifuge tube 3-5000 x g 5 min (~5000 rpm in a Sorvall SS-34 rotor)

        4. Discard supernatant and add 0.4 volume (ie of original volume, here it is 40 ml) TfbI, resupend and ice 15 min.

        5. Pellet cells as in #3.

        6. Discard supernatant and resupend in 0.04 volume TfbII, ice 15 min and either use immediately or quick freeze at -70C for storage. I usually save these in 0.25 to 0.5 ml aliquots. Quick freeze in ethanol-dry ice or liquid nitrogen prior to storage in a -70 to -80 C freezer. Thaw on ice just before using in a transformation experiment.

        I typically transform 50 ul cells with 2-10 ul of a ligation reaction, and you should get between 1x10exp8 to 1x10exp9 cfu's/ug DNA.


        Medium and Buffers

         

        Psi broth (per liter)
        compound amount
        Bacto yeast extract 5 g
        Bacto Tryptone 20 g
        magnesium sulfate 5 g
        pH 7.6 with potassium hydroxide
      •  

         

         

        RETURN TO CONTENT

        [NextPage]

        1. Inoculate from an overnight grown in LB.

        2. Grow in 250 ml "SOB" at 18C until OD600 = 0.6.

        2. On ice for 10 minutes.

        3. Spin at 2500 x g (5000 rpm in a Sorvall GSA or 3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C.

        4. Resuspend cells gently in 80 ml of ice cold "TB". 5. On ice for 10 minutes.

        6. Spin at 2500 x g (5000 rpm in a Sorvall GSA, 5500 rpm in a Sorvall SS-34, or 3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C.

        7. Resuspend cells gently in 20 ml of ice cold "TB".

        8. Add DMSO to a final concentration of 7%.

        9. Place on ice for 10 minutes.

        10. Aliquot into 1-2 ml and freeze in liquid nitrogen.

        11. Store in liquid nitrogen.

         

         

         

        RETURN TO CONTENT

        [NextPage]

        Cosmid Cloning: Cell preparation, DNA packaging, and Cell Transfection

        Protocol taken from Stratagene's Gigapack packaging extracts instruction manual

         

        RETURN TO CONTENT

        [NextPage]

         

         

        RETURN TO CONTENT

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