Team:AUC Turkey/Procedures

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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