|
|
| Line 10: |
Line 10: |
| | <h4> Reagents </h4> | | <h4> Reagents </h4> |
| | <ol> | | <ol> |
| - | <li>LB broth, pH 7</li> | + | <li>HEPES (N-[2-hydroxyethyl] piperazine N'[2-ethane-sulfonic acid])</li> |
| - | <li>10g tryptone</li> | + | <li>Sorbitol</li> |
| - | <li>5g yeast extract</li> | + | <li>Percoll</li> |
| - | <li>5g NaCl</li> | + | <li>PEG 6000</li> |
| - | <li>20% sucrose (autoclaved)</li> | + | <li>Ficoll</li> |
| - | <li>Triton X-100</li> | + | <li>Bovine serum albumin (BSA)</li> |
| - | <li>500mM EDTA stock (pH 8.0)</li> | + | <li>Isoascorbic acid</li> |
| - | <li>Tris-HCl 50mM</li> | + | <li>Sodium pyrophosphate</li> |
| - | <li>NaCl 3M</li> | + | <li>Glutathione</li> |
| - | <li>Isopropanol</li> | + | <li>MgCl2.6H2O</li> |
| - | <li>Autoclaved Milli-Q water</li> | + | <li>MnCl2</li> |
| | + | <li>Sodium-EDTA</li> |
| | </ol> | | </ol> |
| | | | |
| - | <br>
| + | Equipment |
| - | <h4> Procedure </h4>
| + | |
| | | | |
| - | <ol>
| |
| - | <li>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.</li>
| |
| - | <li>Centrifuge 1.5 ml of a 16-18hr bacterial culture for 1 min at 11,500 x g in a polypropylene centrifuge tube.</li>
| |
| - | <li>Remove supernatant by aspirating, leaving the pellet as dry as possible.</li>
| |
| - | <li>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.</li>
| |
| - | <li>Add 50µL of freshly prepared lysozyme solution (10mg/mL in 10mM Tris HCl, pH8), mix by inverting 3X. Lysozyme digests bacterial cell wall.</li>
| |
| - | <li>Immediately incubate at 100oC for 10, 20, 40, 80s.</li>
| |
| - | <li>Spin for 10min 11,500Xg at room temp.</li>
| |
| - | <li>Remove pellet with sterile forceps.</li>
| |
| - | <li>Add to supernatant, 50µL of cold 3M NaAc and 420µL of cold isopropanol.</li>
| |
| - | <li>Incubate 30min at -20oC.</li>
| |
| - | <li>Centrifuge 15min for 11,500Xg at 4oC.</li>
| |
| - | <li>Decant supernatant, invert and drain on clean paper towel.</li>
| |
| - | <li>Add 15µL of cold/4oC TE buffer (0.05M Tris, 0.01M EDTA, pH8).</li>
| |
| - | <li>Incubate for 1hr at 4oC in dark. </li>
| |
| - | <li>Run a small amount of this sample on gel electrophoresis on 0.7% (w/v0) agarose. With the rest, submit to further purification.</li>
| |
| - |
| |
| - | </ol>
| |
| - | <br>
| |
| - | <h4>Further purification (Plasmid from putida)</h4>
| |
| - |
| |
| - | <ol>
| |
| - | <li>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.</li>
| |
| - |
| |
| - | <li>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.</li>
| |
| - |
| |
| - | <li>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.</li>
| |
| | | | |
| | </html> | | </html> |
| | }} | | }} |
This protocol will purify plasmids from bacteria, though identification of plasmids will need to be confirmed by PCR.
Reagents
- HEPES (N-[2-hydroxyethyl] piperazine N'[2-ethane-sulfonic acid])
- Sorbitol
- Percoll
- PEG 6000
- Ficoll
- Bovine serum albumin (BSA)
- Isoascorbic acid
- Sodium pyrophosphate
- Glutathione
- MgCl2.6H2O
- MnCl2
- Sodium-EDTA
Equipment
"
