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> | ||
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<ul><li>In a steril environment, the tare of the container should be measured and subtracted from the overall weight.</li> | <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>7 grams of LB Broth is put in the container.</li> | ||
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<h2>Procedures for Isolation</h2> | <h2>Procedures for Isolation</h2> | ||
- | < | + | <ul><li>The LB Media should be transferred to 1.5 ml centrifuge tubes.</li> |
- | + | <li>These tubes are then centrifuged at 13,000 rpm for 10 minutes at room temperature.</li> | |
- | + | <li>After the centrifuge, the supernatent should be disposed without taking any pellets along with it.</li> | |
- | + | <li>The pelleted cells should be suspended in 250 ul Resuspension Solution and the tubes should be vortexed so that no cell clumps remain.</li> | |
- | + | <li>350 ul Neutralization Solution should be added and the tube should be inverted immediately and throughly by inverting 4-6 times.</li> | |
- | + | <li>Centrifuge for 5 minutes to pellet cell debris and chromosomal DNA.</li> | |
- | + | <li>Transfer the supernatent to a spin column without taking any of the pellets.</li> | |
- | + | <li>Centrifuge the spin column for 1 minutes and discard the liquid at the bottom. Place the column at the same tube again.</li> | |
- | + | <li>Add 500 ul Wash Solution and centrifuge for 30-60 seconds. Discard the flow-through and place column back in.</li> | |
- | + | <li>Repeat the same process again with 500 ul Wash Solution.</li> | |
- | + | <li>Centrifuge for an additional 1 minute.</li> | |
- | + | <li>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.</li> | |
- | + | <li>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.</li> | |
- | + | <li>Discard the spin column and store at -20 C.</li></ul> | |
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<h2>Digestion Protocol</h2> | <h2>Digestion Protocol</h2> | ||
- | < | + | <ul> |
- | + | <li>Take the average of the nucleic acid concentrations measured by the spectrometer.</li> | |
- | + | <li>Divide 500 by the DNA average.</li> | |
- | + | <li>Add 5ul Ne Buffer.</li> | |
- | + | <li>Add 0.5ul BSA Buffer.</li> | |
- | + | <li>Add 1 ul of the enzymes with barrier tips.</li> | |
- | + | <li>If you cut with EcoR1 and SpI, it will be up stream.</li> | |
- | + | <li>If you cut with Xbal and Pst1, it will be down stream.</li> | |
- | + | <li>Subtract the amount of DNA from 42.5 ul. This result will be the amount of NFW used.</li> | |
- | + | <li>Add the NFW with barrier tips and do one pippetting while taking the NFW.</li> | |
- | + | <li>Then the DNA is put into the PCR and is left there for 30 minutes.</li></ul> | |
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<h2>Ligation Protocol</h2> | <h2>Ligation Protocol</h2> | ||
- | < | + | <ul> |
- | + | <li>2ul up stream is put into a eppendorf.</li> | |
- | + | <li>2ul down stream is also added.</li> | |
- | + | <li>2ul plasmid is mixed in as well.</li> | |
- | + | <li>2ul Taq Buffer is inserted to the mixture.</li> | |
- | + | <li>1ul T4 DNA ligase is then added with barrier tips.</li> | |
- | + | <li>11ul NFW is added with barrier tips and should be pipetted once.</li> | |
- | + | <li>Then the DNA is put into the PCR and is left there for 30 minutes.</li> | |
- | </ | + | </ul> |
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<h2>Gel Preparation</h2> | <h2>Gel Preparation</h2> | ||
- | < | + | <ul> |
- | + | <li>Mix 100ml TAE and 0,8 gram agarose in a glass beaker.</li> | |
- | + | <li>The mixture is then heated in a microwave for 3 minutes.</li> | |
- | + | <li>Afterwards, 3,6 ul EtBr is added and the beaker is mixed on a magnetic mixer.</li> | |
- | + | <li>Mold them and wait for 20 minutes fort he gel to harden.</li> | |
- | </ | + | </ul> |
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<h2>Electrophoresis Protocol</h2> | <h2>Electrophoresis Protocol</h2> | ||
- | < | + | <ul> |
- | + | <li>Put 3 ul coloring agent on a strand of parafilm.</li> | |
- | + | <li>Take 7 ul plasmid and do pipeting with the colouring agent.</li> | |
- | + | <li>Switch the pipette to 10 ul and take the colored plasmid.</li> | |
- | + | <li>Place the plasmid into one of the holes of our gel.</li> | |
- | + | <li>Give electricity to the anode and cathode in required amounts.</li> | |
- | + | <li>Wait according to the tank and the amount of electricity.</li> | |
- | + | <li>Use the UV camera to get the results.</li> | |
- | </ | + | </ul> |
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Revision as of 21:28, 16 June 2013