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Team:Grinnell/Notebook/Protocols
From 2011.igem.org
(Difference between revisions)
| Line 13: | Line 13: | ||
<h2><a name="Transformation"></a>Plasmid Transformation</h2> | <h2><a name="Transformation"></a>Plasmid Transformation</h2> | ||
<ol> | <ol> | ||
| - | <li>Thaw | + | <li>Thaw 100μL aliquots of competent cells on ice.</li> |
| - | <li>Add | + | <li>Add 10μL DNA to cells.</li> |
<li>Incubate tubes on ice for 30min.</li> | <li>Incubate tubes on ice for 30min.</li> | ||
<li>Incubate tubes at 42° C for 90sec.</li> | <li>Incubate tubes at 42° C for 90sec.</li> | ||
<li>Incubate tubes on ice for 2min.</li> | <li>Incubate tubes on ice for 2min.</li> | ||
| - | <li>Add | + | <li>Add 300μL LB to cells and incubate shaking at 37° C for 1hr.</li> |
<li>Spread cells on selective media</li> | <li>Spread cells on selective media</li> | ||
<li>Incubate plates overnight at 37° C.</li> | <li>Incubate plates overnight at 37° C.</li> | ||
</ol> | </ol> | ||
| - | + | <h2><a name="GeneReleaser"></a>Isolation of DNA for Colony PCR</h2> | |
| - | <h2><a name=""></a></h2> | + | <a href="http://www.bioventures.com/products.php?cid=10005">GeneReleaser</a> is a proprietary reagent that releases DNA from cells while sequestering cell lysis products that might inhibit DNA polymerases. |
<ol> | <ol> | ||
| - | <li></li> | + | <li>Resuspend the GeneReleaser through inversion, not vortexing. Add 20μL GeneReleaser to each PCR tube.</li> |
| - | <li></li> | + | <li>Add cells from plates with a sterile pipette tip OR 10μL from overnight liquid culture.</li> |
| - | <li></li> | + | <li>Run PCR tubes on following thermal cycle program:</li> |
| - | <li></li> | + | <li><table frame="void" rules="none"> |
| + | <tr><th>Temperature (°C)</th><th>Time (s)</th></tr> | ||
| + | <tr><td>65</td><td>30</td></tr> | ||
| + | <tr><td>8</td><td>30</td></tr> | ||
| + | <tr><td>65</td><td>90</td></tr> | ||
| + | <tr><td>97</td><td>180</td></tr> | ||
| + | <tr><td>8</td><td>60</td></tr> | ||
| + | <tr><td>65</td><td>180</td></tr> | ||
| + | <tr><td>97</td><td>60</td></tr> | ||
| + | <tr><td>65</td><td>60</td></tr> | ||
| + | <tr><td>80</td><td>hold</td></tr></table> | ||
| + | </li> | ||
</ol> | </ol> | ||
| - | + | ||
</html> | </html> | ||
Revision as of 21:11, 9 June 2011
Competent Cells
- Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5 x 10^8 cells/mL, OD600 = 0.3-0.5).
- Chill cells on ice for 15-120min.
- Pellet cells in a prechilled sterile centrifuge tube by centrifugation at 5-8krpm for 5min at 4° C. Discard supernatant.
- Completely resuspend cells in 20mL cold 100mM CaCl2 (10% glycerol), and incubate on ice for 3hr.
- Harvest cells by cetrifugation. Discard supernatant.
- Gently resuspend cells in 0.5mL cold 100mL CaCl2 (10% glycerol). Incubate on ice for at least 1hr and freeze at -80° C.
Plasmid Transformation
- Thaw 100μL aliquots of competent cells on ice.
- Add 10μL DNA to cells.
- Incubate tubes on ice for 30min.
- Incubate tubes at 42° C for 90sec.
- Incubate tubes on ice for 2min.
- Add 300μL LB to cells and incubate shaking at 37° C for 1hr.
- Spread cells on selective media
- Incubate plates overnight at 37° C.
Isolation of DNA for Colony PCR
GeneReleaser is a proprietary reagent that releases DNA from cells while sequestering cell lysis products that might inhibit DNA polymerases.- Resuspend the GeneReleaser through inversion, not vortexing. Add 20μL GeneReleaser to each PCR tube.
- Add cells from plates with a sterile pipette tip OR 10μL from overnight liquid culture.
- Run PCR tubes on following thermal cycle program:
Temperature (°C) Time (s) 65 30 8 30 65 90 97 180 8 60 65 180 97 60 65 60 80 hold
