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• Defining Objectives
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Protocols

Safety

This protocol will purify plasmids from bacteria, though identification of plasmids will need to be confirmed by PCR.

Reagents

1. LB broth, pH 7
2. 10g tryptone
3. 5g yeast extract
4. 5g NaCl
5. 20% sucrose (autoclaved)
6. Triton X-100
7. 500mM EDTA stock (pH 8.0)
8. Tris-HCl 50mM
9. NaCl 3M
10. Isopropanol
11. Autoclaved Milli-Q water

Procedure

1. Inoculate a loopful of Pseudomonas sp. at 25oC, in 10mL LB broth (10g tryptone; 5g yeast extract, 5g NaCl, 1000mL distilled H2O, pH 7.0), and incubate for 16-18hr.
2. Centrifuge 1.5 ml of a 16-18hr bacterial culture for 1 min at 11,500 x g in a polypropylene centrifuge tube.
3. Remove supernatant by aspirating, leaving the pellet as dry as possible.
4. Add to each tube, 400µL of 8% sucrose, 5.0% Triton X-100, 50 mM EDTA, and 10mM Tris HCI (pH 8.0). Mix well.
5. Add 50µL of freshly prepared lysozyme solution (10mg/mL in 10mM Tris HCl, pH8), mix by inverting 3X. Lysozyme digests bacterial cell wall.
6. Immediately incubate at 100oC for 10, 20, 40, 80s.
7. Spin for 10min 11,500Xg at room temp.
8. Remove pellet with sterile forceps.
9. Add to supernatant, 50µL of cold 3M NaAc and 420µL of cold isopropanol.
10. Incubate 30min at -20oC.
11. Centrifuge 15min for 11,500Xg at 4oC.
12. Decant supernatant, invert and drain on clean paper towel.
13. Add 15µL of cold/4oC TE buffer (0.05M Tris, 0.01M EDTA, pH8).
14. Incubate for 1hr at 4oC in dark.
15. Run a small amount of this sample on gel electrophoresis on 0.7% (w/v0) agarose. With the rest, submit to further purification.

Further purification (Plasmid from putida)

1. Layer the resuspended DNA on a 2.5mL bed of saturated CsCl in a polymer tube. Centrifuge for 14hr at 14 000 rpm in a fixed-angle 30 rotor at 2°C. After the run, ~25mL of liquid can be discarded from the top without disturbing the remainder. Mix the lower part to form the concentrated lysate.
2. Slowly dissolve ~5.7g CsCl to the concentrated lysate, until the refractive index is 1.399. Mix solution with Syber-safe or gel-red. Centrifuge for 40hr in a Spinco fixed-angle rotor 50 at 105 000 x g at 12°C. After this run, 2 well-separated bands should be able to be seen. Alternatively, do only this spin where DNA mixed with CsCl is concentrated to a refractive index of 1.399 with the fluorescent DNA stain.
3. Because the DNA was infused with dye, 2 well-separated bands will appear. The upper band is linear and non-circular DNA (junk). The lower band is the plasmid of interest. Remove the upper layer with a micropipette. After it is removed, pool bands from several tubes centrifuge again SW50.1 rotor for 20h at 40 000rpm. Again there will be 2 bands and the lower band is the desired intact plasmid.