Team:AUC Turkey/Procedures

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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