Cloning of catA gene of Acinetobacter calcoaceticus strain ADP1 to observe its expression in E.coli.
On the following day ,Plate number 2 had one single colony which was transformed.In control plate with no Amp,there were thousands of colonies grown.Where are in the rest of the plates there was no growth observed. In plates with AMP SOC 400ul (PSB1A7#3) DNA-1 ~1696 colonies were observed and in 440ul SOC AMP (PSB1A7#4)DNA-2 ~ 2000 colonies were observed.
From the plate-2 ,where there was one colony which was transformed,we took a loop full of bacterial sample and inoculated in LB broth and incubated it overnight and other plates were stored on freezer.
That day we made glycerol stock of sample (AMP SOC 400ul DNA -2) PSb1A7#3 and PSB1A7#4 with 680ml of the sample and 50% of 320ml of the glycerol and stored it in -80 C. After making the glycerol stock we did the gene-JET plasmid mini prep step.Certain few initial steps were not written in the card to follow,those steps are as follows :- A) we tool 3 eppendorf tubes and added 2ml of our sample and centrifuged it ,taking the pelleted part and discarding the supernatant. B) doing it twice ,so that we can get the maximum amount of the plasmid DNA sample.
Now after this we followed the protocols which were given in the mini prep. kit and after doing it we stored the plates in freezer.