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Large scale M13RF isolation

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Large scale M13RF isolation

Double-stranded M13RF is isolated for use in M13 SmaI cut, dephosphorylated vector preparation, described below. The growth conditions of M13-infected bacterial cells (see Figure 1) appears convoluted, but result in a maximal amount of M13 RF molecules per cell. After the M13RF containing bacterial cells are harvested by centrifugation, the double-stranded molecules are isolated using the cesium chloride method for large scale plasmid isolation, as described above. This briefly entailed alkaline cell lysis, sodium acetate precipitation of detergent solubilized proteins and membranes, polyethylene glycol DNA precipitation, and extraction of ethidium bromide stained DNA from a cesium chloride gradient after ultracentrifugation. After removal of the ethidium bromide on an ion-exchange column, the DNA containing fractions are detected by A260 measurement and pooled, and the DNA is concentrated by ethanol precipitation and assayed by restriction enzyme digestion and agarose gel electrophoresis.

Protocol

1. Prepare an early log phase culture of JM101 by inoculating an Ehrlenmeyer flask containing 50 ml of 2xTY with a glycerol stock of JM101 and pre-incubating for 1 hour in a 37degC water bath, with no shaking. Pick a plaque representing the desired M13 clone into four 1.5 ml aliquot of early log phase JM101, and incubate according to the procedure displayed in Figure 1 to result in 4 liters of M13-infected bacteria.

2. Harvest the cells by centrifugation at 7000 rpm for 20 minutes in 500 ml bottles in the RC5-B using the GS3 rotor. Resuspend the cell pellets in fresh 2xTY media to remove contaminating extracellular phage and transfer to two bottles, centrifuge as before, and decant the media. The cell pellets can be frozen at -70degC at this point.

3. Resuspend the cell pellets in a total of 120 ml (30 ml for each bottle) of 1X STB buffer by gently teasing the pellet with a spatula. Add a total 24 ml of lysozyme solution (6 ml for each bottle), gently mix, and incubate for 5 minutes in an ice-water bath.

4. Add 48 ml of 50:2:10 TTE buffer (12 ml for each bottle) and 2 ml of RNase A (10 mg/ml) (0.5 ml for each bottle), gently mix, and incubate in an ice-water bath for 5 minutes.

5. Clear the lysate of precipitated SDS, proteins, membranes, and chromosomal DNA by pouring through a double-layer of cheesecloth. Transfer the lysate into 250 ml centrifuge bottle, centrifuge at 10,000 rpm for 30 minutes at 4deg C in the RC5-B using the GSA rotor.

6. Add 6 ml of 5 mg/ml ethidium bromide, and cesium chloride such that the final concentration of cesium chloride is 1 g/ml.

7. Transfer the sample into 35 ml polyallomer centrifuge tubes and top off with a 1:1 solution of 100:10 TE buffer and cesium chloride, remove air bubbles, seal with rubber stoppers, and crimp properly.

8. Centrifuge at 60,000 rpm to 16-20 hours at 15-20degC in the Sorvall OTD-75B ultracentrifuge using the T-865 rotor.

9. Visualize the ethidium bromide stained DNA under long-wave UV light, and remove the lower DNA band using a 5 cc syringe and a 25 gauge needle. (It may be helpful to remove and discard the upper band first).

10. To remove the ethidium bromide, load the DNA sample onto an 1.5 ml Dowex AG (BioRad) column, equilibrated as before, and collect 0.5 ml fractions.

11. Pool fractions with an A260 of 1.00 or greater into 35 ml Corex glass tubes, add one volume of ddH2O, and ethanol precipitate by adding 2.5 volumes of cold 95% ethanol. Incubate at least 2 hours at -20degC, centrifuge at 10,000 rpm for 45 minutes in the RC5-B using the SS-34 rotor. Gently decant the supernatant, add 80% ethanol, centrifuge as before, decant, and dry the DNA pellet in a vacuum oven.

12. Resuspend the DNA in 10:0.1 TE buffer.

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