Calgary/protocols
From 2007.igem.org
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<li><a href="#transformation" title="Bacterial Transformation Protocol"> Bacterial Transformation </a></li> | <li><a href="#transformation" title="Bacterial Transformation Protocol"> Bacterial Transformation </a></li> | ||
<li><a href="#rehydration" title="Protocol for rehydrating cells from registery"> Rehydration </a></li> | <li><a href="#rehydration" title="Protocol for rehydrating cells from registery"> Rehydration </a></li> | ||
| + | <li><a href="#pcr" title="Protocol for pcr"> PCR </a></li> | ||
</ul> | </ul> | ||
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</p> | </p> | ||
</div> | </div> | ||
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| + | <div style="margin-bottom:60px"> | ||
| + | <p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="pcr"> PCR Protocol </a> </p> | ||
| + | <table width="90%" border="1px" style="margin-bottom:15px;"> | ||
| + | <tr> | ||
| + | <td><b>Reagent</b></td> | ||
| + | <td><b>Volume ( 1x )</b></td> | ||
| + | <td><b>Volume ( 3x )</b></td> | ||
| + | <td><b>Volume ( 5x )</b></td> | ||
| + | <td><b>Volume ( 15x )</b></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Sterile H2O</td> | ||
| + | <td>36 ul</td> | ||
| + | <td>108 ul</td> | ||
| + | <td>180 ul</td> | ||
| + | <td>540 ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>10X Taq Buffer</td> | ||
| + | <td>5 ul</td> | ||
| + | <td>15 ul</td> | ||
| + | <td>25 ul</td> | ||
| + | <td>75 ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>2mM dNTPs</td> | ||
| + | <td>5 ul</td> | ||
| + | <td>15 ul</td> | ||
| + | <td>25 ul</td> | ||
| + | <td>75 ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Forward Primer (100 ug/ul) </td> | ||
| + | <td>1 ul</td> | ||
| + | <td>3 ul</td> | ||
| + | <td>5 ul</td> | ||
| + | <td>15 ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Reverse Primer (100 ug/ul) </td> | ||
| + | <td>1 ul</td> | ||
| + | <td>3 ul</td> | ||
| + | <td>5 ul</td> | ||
| + | <td>15 ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>50mM MgCl2</td> | ||
| + | <td>1.5 ul</td> | ||
| + | <td>4.5 ul</td> | ||
| + | <td>7.5 ul</td> | ||
| + | <td>22.5 ul</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Taq Polymerase (50 ug/ul)</td> | ||
| + | <td>0.5 ul</td> | ||
| + | <td>1.5 ul</td> | ||
| + | <td>2.5 ul</td> | ||
| + | <td>7.5 ul</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <p><b> Thermocycler Conditions </b></p> | ||
| + | <ul> | ||
| + | <li> 1 Cycle - 6 minutes at 95 degrees Celsius </li> | ||
| + | <li> 36 cycles | ||
| + | <ul> | ||
| + | <li> 1 minute at 95 degrees Celsius </li> | ||
| + | <li> 1 minute at 58 degrees Celsius ( this step done at 65 degrees Celsius for higher GC content ) </li> | ||
| + | <li> 1 minute at 72 degrees Celsius </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li> 1 Cycle - 10 minutes at 72 degrees Celsius then HOLD at 4 degrees Celsius </li> | ||
| + | </ul> | ||
| + | <p> Conditions were varied as needed. For example in cases of longer products all 1 minute times were increased to 1.5 to 3 minutes</p> | ||
| + | |||
| + | </div> | ||
| + | |||
| + | |||
</body> | </body> | ||
</html> | </html> | ||
Revision as of 18:58, 12 October 2007
- Thaw 100 ul of competen cells (per transformation) on ice just before they are needed
- Add DNA (max 20ul) thawed cells and mix by flicking the side of the tube. Leave on ice for 30 minutes
- Heat shock for 2 minutes at 42 degrees Celsius or 5 minutes at 37 degrees Celsius
- Place on Ice for 5 minutes
- Add 250ul SOC medium to each tube
- Incubate for 30 to 60 minutes with shaking at 37 degrees Celsius. (Note that for Kanamycin containing plasmides always use one hour)
- Spin down to remove all supernatant except approximately 100 ul
- Plate approximately 30 ul on each of two antibiotic plates
- Grow overnight at 37 degrees Celsius
For this protocol we used three controls
- Positive Control - pBluescript in TOP10 cells on amp resistant plates
- Negative Control - TOP10 cells grown on amp resistant plates
- Negative Control - DB31 cells on amp resistant plates
Biobrick parts are shipped from the registry in a dehydrated from. As such they must be rehydrated before they can be used.
- Puncture a hole through the foil with a pipette tip into the well that corresponds to the Biobrick - standard part that you want
- Add 15 ul of diH20 (deionized water)
- Take 1 ul DNA and transform into your desired competent cells, plate out onto a plate with the correct antibiotic and grow overnight. Your goal here is to obtain single colonies
For this protocol we used three controls
- Positive Control - pBluescript in TOP10 cells on amp resistant plates
- Negative Control - TOP10 cells grown on amp resistant plates
- Negative Control - DB31 cells on amp resistant plates
| Reagent | Volume ( 1x ) | Volume ( 3x ) | Volume ( 5x ) | Volume ( 15x ) |
| Sterile H2O | 36 ul | 108 ul | 180 ul | 540 ul |
| 10X Taq Buffer | 5 ul | 15 ul | 25 ul | 75 ul |
| 2mM dNTPs | 5 ul | 15 ul | 25 ul | 75 ul |
| Forward Primer (100 ug/ul) | 1 ul | 3 ul | 5 ul | 15 ul |
| Reverse Primer (100 ug/ul) | 1 ul | 3 ul | 5 ul | 15 ul |
| 50mM MgCl2 | 1.5 ul | 4.5 ul | 7.5 ul | 22.5 ul |
| Taq Polymerase (50 ug/ul) | 0.5 ul | 1.5 ul | 2.5 ul | 7.5 ul |
Thermocycler Conditions
- 1 Cycle - 6 minutes at 95 degrees Celsius
- 36 cycles
- 1 minute at 95 degrees Celsius
- 1 minute at 58 degrees Celsius ( this step done at 65 degrees Celsius for higher GC content )
- 1 minute at 72 degrees Celsius
- 1 Cycle - 10 minutes at 72 degrees Celsius then HOLD at 4 degrees Celsius
Conditions were varied as needed. For example in cases of longer products all 1 minute times were increased to 1.5 to 3 minutes
