Team:AUC TURKEY/Procedures
From 2013hs.igem.org
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<h2>Procedures for LB Broth Preparation</h2> | <h2>Procedures for LB Broth Preparation</h2> | ||
- | <p> | + | <p><h2>Procedures for LB Agar Preparation</h2> |
- | + | <ul><li>In a steril environment, the tare of the container should be measured and subtracted from the overall weight.</li> | |
- | + | <li>7 grams of LB Broth is put in the container.</li> | |
- | + | <li>200 ml distilled water or is put into a graduated cylinder.</li> | |
- | + | <li>These two are mixed in a beaker.</li> | |
- | + | <li>The opening of the beaker is covered with aliminium folio in such a way that it does not conctact with air.</li> | |
- | + | <li>Autoclave tape is sticked on to the aliminium.</li> | |
- | + | <li>The beaker is placed in to the autoclave machine.</li> | |
- | + | <li>Distilled water or demineralized water is put into the autoclave machine. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker.</li> | |
- | + | <li>After closing the lid of the machine, the 90 minute autoclave process is given start.</li> | |
+ | <li>Take out the beaker and add antibiotics if required.</li> | ||
+ | </ul> | ||
- | + | <i>Warnings for the Autoclaw!</i> | |
- | + | <ul> | |
- | + | <li>Use only demineralised or disttiled water with the device.</li> | |
- | + | <li>Do not open the cover until the manometer drops to zero during the operation.</li> | |
- | + | <li>Please do not use the autoclave for other purposes than sterilization and agar.</li> | |
- | + | <li>Please do not use the autoclave to sterilize explosive, inflammable and oxidizing materials.</li> | |
- | + | <li>Please be cautious when you are closing the lid not to trap your hand.</li> | |
- | + | <li>Please beware of the steam exhaust when you are opening to autoclave after sterilization.</li> | |
+ | <li>Please wear protective gloves before removibg materials from the chamber. Do not access the chamber unless the vapor exhaust is finalized.</li> | ||
+ | </ul> | ||
</div> | </div> | ||
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- | <h2> | + | <h2>Procedures for Transformation</h2> |
- | < | + | <ul><li>Transfer 500 ul LB Broth to 1.5 ml microcentrifuge tubes. This should be done close to a source of fire to prevent contamination.</li> |
- | + | <li>Place the microcentrifuge tubes containing LB Broth in a 42 C heat block for incubation.</li> | |
- | + | <li>Take 1 ul plasmid and place them in 1.5 ml centrifuge tubes.</li> | |
- | + | <li>Add 50 ul competent cells to the plasmid.</li> | |
- | + | <li>Centrifuge them at 3000 rpm for 20-30 seconds.</li> | |
- | + | <li>Incubate the cells in ice for 45 minutes.</li> | |
- | + | <li>After 45 minutes, heat the tubes in the 42 C heat block for a maximum of 90 seconds.</li> | |
- | + | <li>The same tubes should be placed in ice and should be incubated for 5 minutes.</li> | |
- | + | <li>Afterwards, 450 ul LB should be added to the cells to complete them to 500 ul.</li> | |
- | + | <li>The microcentrifuge tubes are then sticked to the shaker horizantally and shaked for 1 hour with 320 rpm at 37 C.</li> | |
- | + | <li>150 ul of the mixture(200 ul for digestion) is then placed on the plate to spread.</li> | |
- | + | <li>It is then spread on the plate and the plates are incubated at 37 C for 16 hours.</li></ul> | |
</div> | </div> | ||
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Revision as of 21:23, 16 June 2013