Team:AUC TURKEY/Procedures
From 2013hs.igem.org
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<ul> | <ul> | ||
- | <li><a href="#tabs-1"> | + | <li><a href="#tabs-1">Procedures for LB Agar Preparation</a></li> |
- | <li><a href="#tabs-2"> | + | <li><a href="#tabs-2">Procedures for LB Broth Preparation</a></li> |
- | <li><a href="#tabs-3"> | + | <li><a href="#tabs-3">Procedures for Transformation</a></li> |
+ | <li><a href="#tabs-4">Procedures for Isolation</a></li> | ||
+ | <li><a href="#tabs-5">Digestion Protocol</a></li> | ||
+ | <li><a href="#tabs-6">Ligation Protocol</a></li> | ||
+ | <li><a href="#tabs-7">Gel Preparation</a></li> | ||
+ | <li><a href="#tabs-8">Electrophoresis Protocol</a></li> | ||
</ul> | </ul> | ||
<div id="tabs-1"> | <div id="tabs-1"> | ||
- | <h2> | + | <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 Agar 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> | ||
<div id="tabs-2"> | <div id="tabs-2"> | ||
- | <h2> | + | <h2>Procedures for LB Broth Preparation</h2> |
- | <p> | + | <p>*In a steril enviroment, the tare of the container should be measured and subtracted from the overall weight. |
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- | *In a steril enviroment, the tare of the container should be measured and subtracted from the overall weight. | + | |
*7 grams of LB Broth is put in the container. | *7 grams of LB Broth is put in the container. | ||
*200 ml distilled water or is put into a graduated cylinder. | *200 ml distilled water or is put into a graduated cylinder. | ||
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*Please be cautious when you are closing the lid not to trap your hand. | *Please be cautious when you are closing the lid not to trap your hand. | ||
*Please beware of the steam exhaust when you are opening to autoclave after sterilization. | *Please beware of the steam exhaust when you are opening to autoclave after sterilization. | ||
- | *Please wear protective gloves before removibg materials from the chamber. Do not access the chamber unless the vapor exhaust is finalized. | + | *Please wear protective gloves before removibg materials from the chamber. Do not access the chamber unless the vapor exhaust is finalized.</p> |
- | + | </div> | |
- | = | + | <div id="tabs-3"> |
- | + | <h2>Content heading 3</h2> | |
- | *Transfer 500 ul LB Broth to 1.5 ml microcentrifuge tubes. This should be done close to a source of fire to prevent contamination. | + | <p>*Transfer 500 ul LB Broth to 1.5 ml microcentrifuge tubes. This should be done close to a source of fire to prevent contamination. |
*Place the microcentrifuge tubes containing LB Broth in a 42 C heat block for incubation. | *Place the microcentrifuge tubes containing LB Broth in a 42 C heat block for incubation. | ||
*Take 1 ul plasmid and place them in 1.5 ml centrifuge tubes. | *Take 1 ul plasmid and place them in 1.5 ml centrifuge tubes. | ||
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*The microcentrifuge tubes are then sticked to the shaker horizantally and shaked for 1 hour with 320 rpm at 37 C. | *The microcentrifuge tubes are then sticked to the shaker horizantally and shaked for 1 hour with 320 rpm at 37 C. | ||
*150 ul of the mixture(200 ul for digestion) is then placed on the plate to spread. | *150 ul of the mixture(200 ul for digestion) is then placed on the plate to spread. | ||
- | *It is then spread on the plate and the plates are incubated at 37 C for 16 hours. | + | *It is then spread on the plate and the plates are incubated at 37 C for 16 hours.</p> |
- | + | </div> | |
- | = | + | <div id="tabs-4"> |
- | + | <h2>Procedures for Isolation</h2> | |
- | *The LB Media should be transferred to 1.5 ml centrifuge tubes. | + | <p>*The LB Media should be transferred to 1.5 ml centrifuge tubes. |
*These tubes are then centrifuged at 13,000 rpm for 10 minutes at room temperature. | *These tubes are then centrifuged at 13,000 rpm for 10 minutes at room temperature. | ||
*After the centrifuge, the supernatent should be disposed without taking any pellets along with it. | *After the centrifuge, the supernatent should be disposed without taking any pellets along with it. | ||
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*Transfer the columns into 1.5 ml microcentrifuge tubes and leave their caps open for 2 minutes so that the ethanol that they contain dissolves in air. | *Transfer the columns into 1.5 ml microcentrifuge tubes and leave their caps open for 2 minutes so that the ethanol that they contain dissolves in air. | ||
*Add 50 ul Elution Buffer to the center of the silica membrane to elute the plasmid DNA. The pipette tip shouldn't the membrane. Incubate for 2 minutes and centrifuge for 2 minutes afterwards. | *Add 50 ul Elution Buffer to the center of the silica membrane to elute the plasmid DNA. The pipette tip shouldn't the membrane. Incubate for 2 minutes and centrifuge for 2 minutes afterwards. | ||
- | *Discard the spin column and store at -20 C. | + | *Discard the spin column and store at -20 C.</p> |
- | + | </div> | |
- | + | <div id="tabs-5"> | |
- | + | <h2>Digestion Protocol</h2> | |
- | + | <p> | |
*Take the average of the nucleic acid concentrations measured by the spectrometer. | *Take the average of the nucleic acid concentrations measured by the spectrometer. | ||
*Divide 500 by the DNA average. | *Divide 500 by the DNA average. | ||
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*Subtract the amount of DNA from 42.5 ul. This result will be the amount of NFW used. | *Subtract the amount of DNA from 42.5 ul. This result will be the amount of NFW used. | ||
*Add the NFW with barrier tips and do one pippetting while taking the NFW. | *Add the NFW with barrier tips and do one pippetting while taking the NFW. | ||
- | *Then the DNA is put into the PCR and is left there for 30 minutes. | + | *Then the DNA is put into the PCR and is left there for 30 minutes.</p> |
- | + | </div> | |
- | = | + | <div id="tabs-6"> |
- | + | <h2>Ligation Protocol</h2> | |
+ | <p> | ||
*2ul up stream is put into a eppendorf. | *2ul up stream is put into a eppendorf. | ||
*2ul down stream is also added. | *2ul down stream is also added. | ||
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*11ul NFW is added with barrier tips and should be pipetted once. | *11ul NFW is added with barrier tips and should be pipetted once. | ||
*Then the DNA is put into the PCR and is left there for 30 minutes. | *Then the DNA is put into the PCR and is left there for 30 minutes. | ||
- | + | </p> | |
- | = | + | </div> |
- | + | <div id="tabs-7"> | |
+ | <h2>Gel Preparation</h2> | ||
+ | <p> | ||
*Mix 100ml TAE and 0,8 gram agarose in a glass beaker. | *Mix 100ml TAE and 0,8 gram agarose in a glass beaker. | ||
*The mixture is then heated in a microwave for 3 minutes. | *The mixture is then heated in a microwave for 3 minutes. | ||
*Afterwards, 3,6 ul EtBr is added and the beaker is mixed on a magnetic mixer. | *Afterwards, 3,6 ul EtBr is added and the beaker is mixed on a magnetic mixer. | ||
*Mold them and wait for 20 minutes fort he gel to harden. | *Mold them and wait for 20 minutes fort he gel to harden. | ||
- | + | </p> | |
- | = | + | </div> |
- | + | <div id="tabs-8"> | |
+ | <h2>Electrophoresis Protocol</h2> | ||
+ | <p> | ||
*Put 3 ul coloring agent on a strand of parafilm. | *Put 3 ul coloring agent on a strand of parafilm. | ||
*Take 7 ul plasmid and do pipeting with the colouring agent. | *Take 7 ul plasmid and do pipeting with the colouring agent. | ||
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*Wait according to the tank and the amount of electricity. | *Wait according to the tank and the amount of electricity. | ||
*Use the UV camera to get the results. | *Use the UV camera to get the results. | ||
+ | </p> | ||
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Revision as of 21:07, 16 June 2013