Team:iTesla-SoundBio/labnotebook/labnotebook060218.html

6/2/18

Mary Elizabeth Adler

Goal: to transform blue coral amilCP into already competent dh5 alpha cells

Materials:

-diluted dh5alpha [shaky]

-0.1M chilled CaCl2 [Chilly]

-1 microcentrifuge tube [on top of Shaky in pipette tip box]

-P1000, P200, P10 micropipettes

-L.B. media [laminar flow hood]

-blue coral amilCP [Frosty]

-Hot water bath [Moisty]

-2 kanamycin plates [laminar flow hood]

-Sterile glass beads [falcon tube laminar flow hood]

 

  1. Turn on water bath to start warming up to 42C so it’s ready when we need it
  2. Label microcentrifuge with group initials and date
  3. Take out competent cells from the incubator [Shaky] and pipette 1500uL of the competent cells into the microcentrifuge tube.
  4. Spin cells down in centrifuge at 4000 rcf for 30 seconds.
  5. Dump out supernatant liquid from tubes, leaving the pellet of cells at the bottom.
  6. Repeat steps 3-5. When adding the second round of competent cells, remember to pipette up and down to resuspend the pellet in liquid.
  7. Pipette 1mL (1000uL) of 0.1M CaCl2 to each tube and pipette up and down to resuspend pellet.
  8. Centrifuge in Grumpy for 30 seconds at 4,000 rcf
  9. Dump out supernatant liquid, leaving the pellets at the bottom.
  10. Pipette 50 ul of CaCl2 to each tube and resuspend pellet.
  11. Pipette 1 ul of the blue coral amil CP into the microcentrifuge tube.
  12. Sit tubes on ice 30 minutes (start at    )
  13. Heat shock tubes in water bath for 45 seconds
  14. Immediately transfer tubes back on ice and let it sit for two minutes
  15. Pipette 500ul  of LB media to each tube
  16. Place tubes in Shaky and incubate for one hour at 37C with shaking.
  17. Under the laminar flow hood, plate the transformation mixtures out on labeled kan plates.
    1. For each plate (two plates, one will be 200ul, the other will be 20ul)
      1. Pour around ten glass beads onto the plate
      2. Pipette full volume of the appreciate transformation mixture onto the plate
      3. Close plate and shake plate from side to side until the beads have spread cells over the majority of the agar surface
      4. Turn over plate and dump beads into waste container filler with bleach (container is covered with foil)
      5. Close plate and set aside
  1. Incubate plates at 37 C overnight in Shaky without shaking
  2. Return materials back to original locations and clean up. Dispose of microcentrifuge tubes that contained the transformation mix. Wash the culture tubes that contained the e.coli cells.