Team:AUC Turkey/Procedures

• 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.
• Afterwards, the

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 values measured by a spectrophotometer to dna.

Divide 500 by DNA average.

Put 5ul Ne Buffer

Put 0.5ul BSA Buffer

Add 1 ul of the enzymas with barrier tips

İf you cut with EcoR1 and SpI, it will be up stream.

İf you cut with Xbal and Pst1, it will be down stream.

(Attention ! enzymes shouldn’t heat)

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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