Team:DTU-Denmarkgogo
From 2014.igem.org
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+ | <pageheader>Protocols</pageheader> | ||
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- | <li> Add one ml sterile 50% glycerol to a cryotube< | + | <li>Add one ml sterile 50% glycerol to a cryotube.</li> |
- | <li>Add one ml overnight culture to a cryotube from step 1< | + | <li>Add one ml overnight culture to a cryotube from step 1</li> |
- | <li>Store at -80 °C< | + | <li>Store at -80 °C</li> |
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+ | </td> | ||
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<th colspan="2" scope="col"><h1>PCR</h1></th> | <th colspan="2" scope="col"><h1>PCR</h1></th> | ||
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+ | <center><a href="h"><img src="https://static.igem.org/mediawiki/2014/8/8e/PCR_PIC.png" width="942" height="180"/> </a> </center> | ||
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+ | <ul> | ||
+ | <li>Make a mastermix of the reagents that are common for all reactions. Make enough for 0,5 extra reaction. Add reagents in the order they are listed above.</li> | ||
+ | <li>The polymerase stock must only be taken out of the freezer when needed and must be put back immediately after use.</li> | ||
+ | <li>Remember to mix thoroughly before aliquotting to separate PCR tubes, and try not to make any bubbles. Reverse pipetting works well for aliquoting.</li> | ||
+ | <li>Mark the PCR tubes on lid and side and place in thermocycler.</li> | ||
+ | <li>The annealing temperature should match the primer melting temperatures. This can be hard as Tm predictors tend to disagree.</li> | ||
+ | <li>Elongation time should be AT LEAST 30 seconds per kb of the longest expected fragment (for the X7 polymerase).</li> | ||
+ | </ul> | ||
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- | <th colspan="2" scope="col">< | + | <th colspan="2" scope="col"><h1>Casting of gels and gel electrophoresis (remember to include EtBr)</h1></th> |
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- | + | <ol> | |
- | + | <li>A</li> | |
- | + | <li>A</li> | |
- | + | <li>S</li> | |
- | + | </ol> | |
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- | <th colspan="2" scope="col">< | + | <th colspan="2" scope="col"><h1>Preparation of Cam stock solution</h1></th> |
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- | + | <ol> | |
- | + | <li>h</li> | |
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- | <th colspan="2" scope="col">< | + | <th colspan="2" scope="col"><h1>Recipe on M9 minimal media</h1></th> |
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- | + | <ol> | |
- | < | + | <li>Make M9 salts</li> |
- | + | <li>To make M9 Salts aliquot 800ml H2O and add</li> | |
- | < | + | <ul> |
- | + | <li>64g Na2HPO4-7H2O</li> | |
- | + | <li>15g KH2PO4</li> | |
+ | <li>2.5g NaCl</li> | ||
+ | <li>5.0g NH4Cl</li> | ||
+ | <li>Stir until dissolved</li> | ||
+ | <li>Adjust to 1000ml with distilled H2O</li> | ||
+ | <li>Sterilize by autoclaving</li> | ||
+ | </ul> | ||
+ | <li>Measure ~700ml of distilled H2O (sterile)</li> | ||
+ | <li>Add 200ml of M9 salts</li> | ||
+ | <li>Add 2ml of 1M MgSO4 (sterile)</li> | ||
+ | <li>Add 20 ml of 20% glucose (or other carbon source)</li> | ||
+ | <li>Add 100ul of 1M CaCl2 (sterile)</li> | ||
+ | <li>Adjust to 1000ml with distilled H2O</li> | ||
+ | |||
+ | </ol> | ||
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- | <th colspan="2" scope="col">< | + | <th colspan="2" scope="col"><h1>Restriction analysis</h1></th> |
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- | < | + | <li>Make a mastermix containing.</li> |
- | + | <ul> | |
- | + | <li>1X NEBuffer (according to the desired restriction enzyme)</li> | |
- | < | + | <li>1X BSA (note if NEBuffer is called X.1 BSA is already added in the buffer)</li> |
- | + | <li>1 unit/μl restriction enzyme</li> | |
- | + | ||
+ | </ul> | ||
+ | <li>Divide the mastermix into tubes and add DNA to a final concentration of 10 ng/μl</li> | ||
+ | <li>Incubate the reaction mix at the restriction enzymes optimal temperature for 30-60 minutes</li> | ||
+ | <li>Add loading buffer to samples and run on 1% agarose gel with ethidium bromide 0.2 μg/ml</li> | ||
+ | <li>Analyze your gel</li> | ||
+ | </ol> | ||
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- | <th colspan="2" scope="col">< | + | <th colspan="2" scope="col"><h1>next</h1></th> |
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+ | </ul> | ||
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- | <th colspan="2" scope="col">< | + | <th colspan="2" scope="col"><h1>Protocol for eating spinach</h1></th> |
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+ | <img style="float: left; "src="http://i.perezhilton.com/wp-content/uploads/2010/03/popeye46704__oPt.jpg" height="300"> </img> | ||
+ | </td> | ||
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Latest revision as of 14:32, 1 August 2014
Glycerol stock-preservation |
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