Haynes Lab:Notebook/Lab Traning

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Pradyumna Kadambi/Undergraduate Lab Traning

DH5α Transformation with KAH013 Plasmid 6/25/2013

  1. In ice bath, 1μl KAH013 plasmid and 50μl competent E.Coli were combined.
  2. Waited 10 minutes for bacteria to accept plasmid.
  3. Transformed bacteria were then added to Agarose gel petri dish, rolling beads were used to evenly spread bacteria.
  4. Petri dish was labeled with initials date and plasmid name("PK 6/25/2013 KAH13") and was placed in incubator at 37 degrees for two days(6/25-6/26).
  5. Resultant colonies were picked by Rene and grown in liquid LB media overnight(6/27/2013).

DH5α Incubation in Liquid Media 6/28/2013

  1. With a pipetter, 3mL of LB+amp was added to a test tube.
  2. A colony was selected on the culture plate(plate was overgrown), and a circle was drawn where the colony was selected.
  3. Micropipette tip was lightly touched to the colony making sure not to scrape any of the agar gel. Micropipette tip was then ejected into test tube.
  4. Test tube was incubated with shaker for 7 hours at 37 degrees.

'DH5α Mini Prep with KAH013 6/28/2013

  • Colonies that Rene had incubated in liquid media on 6/27 were used.
  1. Using a micropipette, a 2mL centrifuge tube was filled with the incubated liquid media.
  2. The solution was centrifuged at 13.1g for 3 minutes (If a pellet has not completely condensed centrifuge again until pellet does condense).
  3. Using a micropipette, the supernatant was removed until 600μL of solution remained in the centrifuge tube. (Discard supernatant into the bleach container in the fume hood)
  4. Pellet was broken up using the vortex.
  5. Added 100μL of lysis buffer to the centrifuge tube and inverted 4-6 times and waited 1 min.
  6. Added 350μL of lysis buffer. Invert the solution until it turns yellow, make sure no blue pockets are left.
  7. Centrifuge the solution at

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