"
Team:Carnegie Mellon/Met-Protocols
From 2012.igem.org
(Difference between revisions)
| Line 124: | Line 124: | ||
<table> | <table> | ||
| - | <tr> <td width = "25"> 1. </td><td width = "200">Wash 1mL induced cells once with PBS and resuspend in 1mL M9 media.</td></tr> | + | <tr> <td width = "25"> 1. </td><td width = "200"> First culture a fresh batch of cells using 1:100 of overnight culture of promoter construct. Culture for around 2 hours or until the solution is lightly turbid. </td></tr> |
| + | <tr><td>2. </td><td> Induce cells by adding 1ul of 1mM IPTG. </td></tr> | ||
| + | <tr><td>3. </td><td> Wash 1mL induced cells once with PBS and resuspend in 1mL M9 media.</td></tr> | ||
| + | <tr><td>4. </td><td title="To increase DFHBI fluorescence"> Add 1µL of 50nM Mg2+ to the tubes (from MgCl2 from PCR kit)</td></tr> | ||
| + | <tr><td>5. </td><td>Aliquot (100ul/200ul) of cells into desired wells of a 96-well plate.</td></tr> | ||
| + | <tr><td>6. </td><td>Add various doses of DFHBI to the wells, followed by adding the desired doses of MG</td></tr> | ||
| + | <tr><td>7. </td><td>Plate read with plate reader (Tecan Safire II). First find cell density using OD600. Then use Excitation/Emission of 469/501 for Spinach and 635/660 for Malachite-green. </td></tr> | ||
| + | </table> | ||
| + | </p> | ||
| + | |||
| + | |||
| + | <p><h2 id = "section1-3"> | ||
| + | <b>Cloning Protocol</b></h2> | ||
| + | |||
| + | <table> | ||
| + | <tr> <td width = "25"> 1. </td><td width = "200">Start digestion of vector and insert DNA using desired restriction enzymes </td></tr> | ||
<tr><td>2. </td><td title="To increase DFHBI fluorescence"> Add 1µL of 50nM Mg2+ to the tubes (from MgCl2 from PCR kit)</td></tr> | <tr><td>2. </td><td title="To increase DFHBI fluorescence"> Add 1µL of 50nM Mg2+ to the tubes (from MgCl2 from PCR kit)</td></tr> | ||
<tr><td>3. </td><td>Aliquot (100ul/200ul) of cells into desired wells of a 96-well plate.</td></tr> | <tr><td>3. </td><td>Aliquot (100ul/200ul) of cells into desired wells of a 96-well plate.</td></tr> | ||
| Line 130: | Line 145: | ||
<tr><td>5. </td><td>Plate read with plate reader (Tecan Safire II). First find cell density using OD600. Then use Excitation/Emission of 469/501 for Spinach and 635/660 for Malachite-green. </td></tr> | <tr><td>5. </td><td>Plate read with plate reader (Tecan Safire II). First find cell density using OD600. Then use Excitation/Emission of 469/501 for Spinach and 635/660 for Malachite-green. </td></tr> | ||
</table> | </table> | ||
| - | |||
</p> | </p> | ||
| - | |||
| - | |||
</html> | </html> | ||
{{:Team:Carnegie_Mellon/Templates/Footer}} | {{:Team:Carnegie_Mellon/Templates/Footer}} | ||
Revision as of 03:29, 17 September 2012
Protocols
Overview
Dosage Curve
| 1. | First culture a fresh batch of cells using 1:100 of overnight culture of promoter construct. Culture for around 2 hours or until the solution is lightly turbid. |
| 2. | Induce cells by adding 1ul of 1mM IPTG. |
| 3. | Wash 1mL induced cells once with PBS and resuspend in 1mL M9 media. |
| 4. | Add 1µL of 50nM Mg2+ to the tubes (from MgCl2 from PCR kit) |
| 5. | Aliquot (100ul/200ul) of cells into desired wells of a 96-well plate. |
| 6. | Add various doses of DFHBI to the wells, followed by adding the desired doses of MG |
| 7. | Plate read with plate reader (Tecan Safire II). First find cell density using OD600. Then use Excitation/Emission of 469/501 for Spinach and 635/660 for Malachite-green. |
Cloning Protocol
| 1. | Start digestion of vector and insert DNA using desired restriction enzymes |
| 2. | Add 1µL of 50nM Mg2+ to the tubes (from MgCl2 from PCR kit) |
| 3. | Aliquot (100ul/200ul) of cells into desired wells of a 96-well plate. |
| 4. | Add various doses of DFHBI to the wells, followed by adding the desired doses of MG |
| 5. | Plate read with plate reader (Tecan Safire II). First find cell density using OD600. Then use Excitation/Emission of 469/501 for Spinach and 635/660 for Malachite-green. |
