Team:iTesla-SoundBio/TransformingintoE.ColiProtocolTemplate

Highlighted text are things that you need to fill in or change for your particular lab notebook depending on the variables, and concentrations.

 

Transformation of _______ Ligation Reactions.

 

Materials:

-diluted dh5alpha cells [shaky, overnight prep step]

-0.1M chilled CaCl2 [Chilly]

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

-P1000, P200, P10 micropipettes

-Ligation Reactions [from previous step]

-L.B. media [laminar flow hood, or SoundBio Storage]

-Hot water bath [Moisty]

-Chlor and amp plates[laminar flow hood]

-Sterile glass beads [falcon tube laminar flow hood]

 

    1. Add 40ul of the overnight culture to 4ml of fresh LB in a new culture tube 1.5 - 2 hours before you start the transformation. Put tube back into incubator with shaking.
      1. Consider how much e.coli to dilute in preparation for the transformation. You will end up using 200ul of the competent dilution for each reaction, but you also will need extra to test in the cuvette reader. So, dilute 1:100 of the overnight culture into 200ul of broth x number of reactions + 1-1.5 ml extra broth. For example, for 9 reactions, we diluted 40ul of the overnight cells in 4 mL of LB broth.

 

  • Turn on water bath to start warming up to 42C so it’s ready when we need it

 

  1. Label microcentrifuge tubes accordingly, so we can be ready down the line (this also serves as a list of reactions to do):
    1.  
  2. About 1-1.25 hours into the incubation period, measure OD600 concentration in the cuvette reader:
    1. Take cuvette out of nanodrop.
    2. Pipette 1000ul of LB broth into the cuvette.
    3. Open the Nanodrop 2000C program and select “Cell Cultures.” Make sure the “cuvette” box is checked.
    4. Put cuvette into nanodrop and select “Blank.”
    5. Rinse cuvette out with diH2O.
    6. Pipette 500 ul of the incubating cells into the cuvette.
    7. Pipette 500 ul of LB broth into the cuvette.
    8. Put cuvette into nanodrop holder, and click “Measure.”
    9. The concentration should be between 0.3 and 0.4. Since we diluted our cells, make sure to double the output reading to get the actual current concentration of the incubating cells.
    10. If the OD600 of the undiluted cells is between 0.3 and 0.4, then take the cells out of the incubator and begin the transformation. If the OD600 reading is not between 0.3 and 0.4, then let cells incubate longer, and check reading again.
  3. Take out competent cells from the incubator.
  4. Transfer 1500 ul to a single microcentrifuge tube (pipette 750 ul at a time).
  5. Spin cells down in centrifuge at 4000 rcf for 30 seconds.
  6. Dump out supernatant liquid from tubes, leaving the pellet of cells at the bottom.
  7. Repeat steps 5-6 until all cells are pelleted.
  8. Pipette 1mL (1000uL) of 0.1M CaCl2 to  the tube and pipette up and down to resuspend the pellet.
  9. Centrifuge in Grumpy for 30 seconds at 4,000 rcf
  10. Dump out supernatant liquid, leaving the pellets at the bottom.
  11. Pipette _____ul of CaCl2 to the  tube and resuspend pellet.
    1. Add 200ul of CaCl2 per transformation reaction. Since you might have too many reactions for the amount of CaCl2 to fit in a 1.5 ul, you might need to resuspend pellet in ~800ul, then transfer solution into a 2ul tube and add remaining needed CaCl2.
  12. Pipette 200 ul of competent cells into each waiting microcentrifuge tube.
  13. Pipette 10 ul of each ligation reaction into the corresponding microcentrifuge tube.
  14. Sit tubes on ice 30 minutes (Time started:       )
  15. Heat shock tubes in water bath for 45 seconds
  16. Immediately transfer tubes back on ice and let it sit for two minutes
  17. Pipette 500ul of LB media to each tube
  18. Place tubes in Shaky and incubate for one hour at 37C with shaking. (started at     )
  19. Under the laminar flow hood, plate the transformation mixtures out on labeled chlor plates. (Label with reaction, date, and 200ul/20ul, around the edges of the plate and on the agar side).
    1. For each plate (two plates per reaction, one will be 200ul, the other will be 20ul)
      1. Pour around ten glass beads onto the plate
      2. Pipette full volume of the appropriate 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.