Team:Calgary/Notebook/Protocols/Process1
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- | This protocol will purify plasmids from bacteria, though identification of plasmids will need to be confirmed by PCR | + | This protocol will purify plasmids from bacteria, though identification of plasmids will need to be confirmed by PCR.</p> |
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Revision as of 08:16, 24 September 2011
Plasmid Extraction
This protocol will purify plasmids from bacteria, though identification of plasmids will need to be confirmed by PCR.
Reagents
- LB broth, pH 7
- 10g tryptone
- 5g yeast extract
- 5g NaCl
- 20% sucrose (autoclaved)
- Triton X-100
- 500mM EDTA stock (pH 8.0)
- Tris-HCl 50mM
- NaCl 3M
- Isopropanol
- Autoclaved Milli-Q water
Procedure
- 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.
- Centrifuge 1.5 ml of a 16-18hr bacterial culture for 1 min at 11,500 x g in a polypropylene centrifuge tube.
- Remove supernatant by aspirating, leaving the pellet as dry as possible.
- 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.
- Add 50µL of freshly prepared lysozyme solution (10mg/mL in 10mM Tris HCl, pH8), mix by inverting 3X. Lysozyme digests bacterial cell wall.
- Immediately incubate at 100oC for 10, 20, 40, 80s.
- Spin for 10min 11,500Xg at room temp.
- Remove pellet with sterile forceps.
- Add to supernatant, 50µL of cold 3M NaAc and 420µL of cold isopropanol.
- Incubate 30min at -20oC.
- Centrifuge 15min for 11,500Xg at 4oC.
- Decant supernatant, invert and drain on clean paper towel.
- Add 15µL of cold/4oC TE buffer (0.05M Tris, 0.01M EDTA, pH8).
- Incubate for 1hr at 4oC in dark.
- 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)
- 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.
- 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.
- 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.