Haynes Lab:Notebook:Shared/Cloning Bootcamp/2013/06/25

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Jan Simper - 06/25/13

  • Learned about plasmids, DNA uptake, bacterial transformation, and the whole process of replicating DNA and ensuring that the DNA sample is the one that is wanted. Then went into the lab and performed bacterial transformation using this protocol:

1. Take 9 ul of ddH2O and put it into a tube with 1 ul of the plasmid DNA (in this case, the DNA was KAH47). Wait for the plasmid DNA to thaw before adding. 2. Mix the contents in the tube by flicking it and then pipette 40 ul of E. coli cells into the tube. Mix the contents again, then pipette the contents onto an agar/LB plate. Minimize the amount of time the lid is open. 3. Use glass beads to spread the plasmid DNA throughout the plate, 5-10 glass beads should be all that is needed. Then put the glass beads into the container with the rest of the dirty glass beads and the ethanol. Make sure the ethanol covers the top layer of beads. 4. Label the agar plate with name, date, plasmid DNA part number, and type of E. coli cell (in this case, DH5 alpha turbo). 5. Place in the incubator.

Learned that E. coli cells will typically enter into a state of slow growth and death around 5-7 hours, so the best time to move on to the next step is after 5-7 hours have elapsed since incubation.





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