Team:AUC TURKEY/Procedures
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+ | == Procedures for LB Agar Preparation == | ||
+ | |||
+ | *In a steril enviroment, the tare of the container should be measured and subtracted from the overall weight. | ||
+ | *7 grams of LB Agar is put in the container. | ||
+ | *200 ml distilled water or is put into a graduated cylinder. | ||
+ | *These two are mixed in a beaker. | ||
+ | *The opening of the beaker is covered with aliminium folio in such a way that it does not conctact with air. | ||
+ | *Autoclave tape is sticked on to the aliminium. | ||
+ | *The beaker is placed in to the autoclave machine. | ||
+ | *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. | ||
+ | *After closing the lid of the machine, the 90 minute autoclave process is given start. | ||
+ | *Take out the beaker and add antibiotics if required. | ||
+ | |||
+ | ''Warnings for the Autoclaw!'' | ||
+ | |||
+ | *Use only demineralised or disttiled water with the device. | ||
+ | *Do not open the cover until the manometer drops to zero during the operation. | ||
+ | *Please do not use the autoclave for other purposes than sterilization and agar. | ||
+ | *Please do not use the autoclave to sterilize explosive, inflammable and oxidizing materials. | ||
+ | *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 wear protective gloves before removibg materials from the chamber. Do not access the chamber unless the vapor exhaust is finalized. | ||
+ | |||
+ | |||
+ | == Procedures for LB Broth Preparation == | ||
+ | |||
+ | *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. | ||
+ | *200 ml distilled water or is put into a graduated cylinder. | ||
+ | *These two are mixed in a beaker. | ||
+ | *The opening of the beaker is covered with aliminium folio in such a way that it does not conctact with air. | ||
+ | *Autoclave tape is sticked on to the aliminium. | ||
+ | *The beaker is placed in to the autoclave machine. | ||
+ | *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. | ||
+ | *After closing the lid of the machine, the 90 minute autoclave process is given start. | ||
+ | *Take out the beaker and add antibiotics if required. | ||
+ | |||
+ | ''Warnings for the Autoclave!'' | ||
+ | |||
+ | *Use only demineralised or disttiled water with the device. | ||
+ | *Do not open the cover until the manometer drops to zero during the operation. | ||
+ | *Please do not use the autoclave for other purposes than sterilization and agar. | ||
+ | *Please do not use the autoclave to sterilize explosive, inflammable and oxidizing materials. | ||
+ | *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 wear protective gloves before removibg materials from the chamber. Do not access the chamber unless the vapor exhaust is finalized. | ||
+ | |||
+ | == Procedures for Transformation == | ||
+ | |||
+ | *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. | ||
+ | *Take 1 ul plasmid and place them in 1.5 ml centrifuge tubes. | ||
+ | *Add 50 ul competent cells to the plasmid. | ||
+ | *Centrifuge them at 3000 rpm for 20-30 seconds. | ||
+ | *Incubate the cells in ice for 45 minutes. | ||
+ | *After 45 minutes, heat the tubes in the 42 C heat block for a maximum of 90 seconds. | ||
+ | *The same tubes should be placed in ice and should be incubated for 5 minutes. | ||
+ | *Afterwards, 450 ul LB should be added to the cells to complete them to 500 ul. | ||
+ | *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. | ||
+ | *It is then spread on the plate and the plates are incubated at 37 C for 16 hours. | ||
+ | |||
+ | == Procedures for Isolation == | ||
+ | |||
+ | *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. | ||
+ | *After the centrifuge, the supernatent should be disposed without taking any pellets along with it. | ||
+ | *The pelleted cells should be suspended in 250 ul Resuspension Solution and the tubes should be vortexed so that no cell clumps remain. | ||
+ | *350 ul Neutralization Solution should be added and the tube should be inverted immediately and throughly by inverting 4-6 times. | ||
+ | *Centrifuge for 5 minutes to pellet cell debris and chromosomal DNA. | ||
+ | *Transfer the supernatent to a spin column without taking any of the pellets. | ||
+ | *Centrifuge the spin column for 1 minutes and discard the liquid at the bottom. Place the column at the same tube again. | ||
+ | *Add 500 ul Wash Solution and centrifuge for 30-60 seconds. Discard the flow-through and place column back in. | ||
+ | *Repeat the same process again with 500 ul Wash Solution. | ||
+ | *Centrifuge for an additional 1 minute. | ||
+ | *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. | ||
+ | *Discard the spin column and store at -20 C. | ||
+ | |||
+ | |||
+ | == Digestion protocol == | ||
+ | |||
+ | *Take the average of the nucleic acid concentrations measured by the spectrometer. | ||
+ | *Divide 500 by the DNA average. | ||
+ | *Add 5ul Ne Buffer. | ||
+ | *Add 0.5ul BSA Buffer. | ||
+ | *Add 1 ul of the enzymes with barrier tips. | ||
+ | *If you cut with EcoR1 and SpI, it will be up stream. | ||
+ | *If you cut with Xbal and Pst1, it will be down stream. | ||
+ | *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. | ||
+ | *Then the DNA is put into the PCR and is left there for 30 minutes. | ||
+ | |||
+ | == Ligation protocol == | ||
+ | |||
+ | *2ul up stream is put into a eppendorf. | ||
+ | *2ul down stream is also added. | ||
+ | *2ul plasmid is mixed in as well. | ||
+ | *2ul Taq Buffer is inserted to the mixture. | ||
+ | *1ul T4 DNA ligase is then added with barrier tips. | ||
+ | *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. | ||
+ | |||
+ | == Gel Preparation == | ||
+ | |||
+ | *Mix 100ml TAE and 0,8 gram agarose in a glass beaker. | ||
+ | *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. | ||
+ | *Mold them and wait for 20 minutes fort he gel to harden. | ||
+ | |||
+ | == Electrophoresis Protocol == | ||
+ | |||
+ | *Put 3 ul coloring agent on a strand of parafilm. | ||
+ | *Take 7 ul plasmid and do pipeting with the colouring agent. | ||
+ | *Switch the pipette to 10 ul and take the colored plasmid. | ||
+ | *Place the plasmid into one of the holes of our gel. | ||
+ | *Give electricity to the anode and cathode in required amounts. | ||
+ | *Wait according to the tank and the amount of electricity. | ||
+ | *Use the UV camera to get the results. | ||
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Revision as of 20:33, 16 June 2013