*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.
*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.
*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.
*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.
*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.
*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.
*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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