User:Samiye Yaman/Notebook/Lab 571/2011/09/20
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Two separate procedures were done.
1)A culture media of E.Coli was prepared for the protein expression.
-0.875 g of Lysogeny Broth and 35 ml of water were added to four separate 250mL flasks and by using a piece of aluminum foil top of the each flask was covered.
-Flasks were placed to autoclave machine for 50 minutes. The temperature was 121 F.
-After being autoclaved for 50 min, they were let to be cooled.
-0.450g of ampicilin was mixed with 4.5 ml of water.
-35uL of the ampicilin mixture was added to each flask.
-By using sterile methods, bacteria was transferred to each flask with ampicilin mixture.
-The flasks were placed in the uncubater overnight. The incubater was at 37°C.
-Online webpage,( http://www.basic.northwestern.edu/biotools/oligocalc.html) was used to have a better understanding of PCR and find the forward and reverse primers necessary for the experiment.
-A solution of 5 uL of 10X Pfu reaction buffer,1 uL of PfuTurbo DNA polymerase,1 uL of dNTP mix,1 uL of template DNA, 1.25 uL of primer 1 and 1.25 uL of primer 2 was prepared.
-Before placing the solution in the thermocycler at 95°C for 30 seconds, 25 uL of BioRad Chill out wax, dark pink, was added to the top of the solution in order to prevent the evaporation of the solution.
-The solution with the wax was kept in the thermocycler, which was at 4°C, for 24 hours.
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