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