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 camera to get the results.

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