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| - | {{AUC_Turkey}}
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| - | == Procedures for LB Agar Preparation ==
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| - | *In a steril enviroment, the tare of the container should be measured and subtracted from the overall weight.
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| - | *7 grams of LB Agar is put in the container.
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| - | *200 ml distilled water or is put into a graduated cylinder.
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| - | *These two are mixed in a beaker.
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| - | *The opening of the beaker is covered with aliminium folio in such a way that it does not conctact with air.
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| - | *Autoclave tape is sticked on to the aliminium.
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| - | *The beaker is placed in to the autoclave machine.
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| - | *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.
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| - | *After closing the lid of the machine, the 90 minute autoclave process is given start.
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| - | *Take out the beaker and add antibiotics if required.
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| - |
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| - | ''Warnings for the Autoclaw!''
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| - | *Use only demineralised or disttiled water with the device.
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| - | *Do not open the cover until the manometer drops to zero during the operation.
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| - | *Please do not use the autoclave for other purposes than sterilization and agar.
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| - | *Please do not use the autoclave to sterilize explosive, inflammable and oxidizing materials.
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| - | *Please be cautious when you are closing the lid not to trap your hand.
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| - | *Please beware of the steam exhaust when you are opening to autoclave after sterilization.
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| - | *Please wear protective gloves before removibg materials from the chamber. Do not access the chamber unless the vapor exhaust is finalized.
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| - | == Procedures for LB Broth Preparation ==
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| - |
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| - | *In a steril enviroment, the tare of the container should be measured and subtracted from the overall weight.
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| - | *7 grams of LB Broth is put in the container.
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| - | *200 ml distilled water or is put into a graduated cylinder.
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| - | *These two are mixed in a beaker.
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| - | *The opening of the beaker is covered with aliminium folio in such a way that it does not conctact with air.
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| - | *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.
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| - | *Please do not use the autoclave to sterilize explosive, inflammable and oxidizing materials.
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| - | *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.
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| - | *Please wear protective gloves before removibg materials from the chamber. Do not access the chamber unless the vapor exhaust is finalized.
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| - |
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| - | == Procedures for Transformation ==
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| - | *Transfer 500 ul LB Broth to 1.5 ml microcentrifuge tubes. This should be done close to a source of fire to prevent contamination.
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| - | *Place the microcentrifuge tubes containing LB Broth in a 42 C heat block for incubation.
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| - | *Take 1 ul plasmid and place them in 1.5 ml centrifuge tubes.
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| - | *Add 50 ul competent cells to the plasmid.
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| - | *Centrifuge them at 3000 rpm for 20-30 seconds.
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| - | *Incubate the cells in ice for 45 minutes.
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| - | *After 45 minutes, heat the tubes in the 42 C heat block for a maximum of 90 seconds.
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| - | *The same tubes should be placed in ice and should be incubated for 5 minutes.
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| - | *Afterwards, 450 ul LB should be added to the cells to complete them to 500 ul.
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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.
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| - | *150 ul of the mixture(200 ul for digestion) is then placed on the plate to spread.
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| - | *It is then spread on the plate and the plates are incubated at 37 C for 16 hours.
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| - | == Procedures for Isolation ==
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| - | *The LB Media should be transferred to 1.5 ml centrifuge tubes.
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| - | *These tubes are then centrifuged at 13,000 rpm for 10 minutes at room temperature.
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| - | *After the centrifuge, the supernatent should be disposed without taking any pellets along with it.
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| - | *The pelleted cells should be suspended in 250 ul Resuspension Solution and the tubes should be vortexed so that no cell clumps remain.
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| - | *350 ul Neutralization Solution should be added and the tube should be inverted immediately and throughly by inverting 4-6 times.
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| - | *Centrifuge for 5 minutes to pellet cell debris and chromosomal DNA.
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| - | *Transfer the supernatent to a spin column without taking any of the pellets.
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| - | *Centrifuge the spin column for 1 minutes and discard the liquid at the bottom. Place the column at the same tube again.
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| - | *Add 500 ul Wash Solution and centrifuge for 30-60 seconds. Discard the flow-through and place column back in.
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| - | *Repeat the same process again with 500 ul Wash Solution.
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| - | *Centrifuge for an additional 1 minute.
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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.
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| - | *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.
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| - | *Discard the spin column and store at -20 C.
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| - | == Digestion protocol ==
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| - | *Take the average of the nucleic acid concentrations measured by the spectrometer.
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| - | *Divide 500 by the DNA average.
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| - | *Add 5ul Ne Buffer.
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| - | *Add 0.5ul BSA Buffer.
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| - | *Add 1 ul of the enzymes with barrier tips.
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| - | *If you cut with EcoR1 and SpI, it will be up stream.
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| - | *If you cut with Xbal and Pst1, it will be down stream.
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| - | *Subtract the amount of DNA from 42.5 ul. This result will be the amount of NFW used.
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| - | *Add the NFW with barrier tips and do one pippetting while taking the NFW.
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| - | *Then the DNA is put into the PCR and is left there for 30 minutes.
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| - | == Ligation protocol ==
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| - | *2ul up stream is put into a eppendorf.
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| - | *2ul down stream is also added.
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| - | *2ul plasmid is mixed in as well.
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| - | *2ul Taq Buffer is inserted to the mixture.
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| - | *1ul T4 DNA ligase is then added with barrier tips.
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| - | *11ul NFW is added with barrier tips and should be pipetted once.
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| - | *Then the DNA is put into the PCR and is left there for 30 minutes.
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| - | == Gel Preparation ==
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| - | *Mix 100ml TAE and 0,8 gram agarose in a glass beaker.
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| - | *The mixture is then heated in a microwave for 3 minutes.
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| - | *Afterwards, 3,6 ul EtBr is added and the beaker is mixed on a magnetic mixer.
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| - | *Mold them and wait for 20 minutes fort he gel to harden.
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| - | == Electrophoresis Protocol ==
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| - | *Put 3 ul coloring agent on a strand of parafilm.
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| - | *Take 7 ul plasmid and do pipeting with the colouring agent.
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| - | *Switch the pipette to 10 ul and take the colored plasmid.
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| - | *Place the plasmid into one of the holes of our gel.
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| - | *Give electricity to the anode and cathode in required amounts.
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| - | *Wait according to the tank and the amount of electricity.
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| - | *Use the UV camera to get the results.
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| - | <forum_subtle/>
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