Transforming Competent Cells - DH5-alpha cells

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1. Thaw the DH5-alpha cells (from -80ºC freezer), plasmid sample (from -20ºC freezer), and the antibiotic stock (from -20ºC freezer) ON ICE.
   
   
2. Split the thawed cells into 2x 100µl aliquots.
   
   
3. To each aliquot add 1µl of plasmid DNA.
   
   
4. Incubate ON ICE for ~30 minutes. Incubation can be longer than this but certainly NO shorter.
   
   
5. During this incubation time prepare the antibiotic agar plates:
   
   
   1. Melt the agar jar for 15 minutes with the microwave on ‘defrost’. Check on the bottle every 5 or so minutes to ensure it is not overflowing.
      
      
   2. Cool the agar bottle in 67ºC water bath. During this time switch on the the laminar flow hood to create a sterile environment.
      
      
   3. Once cool take the agar and petri dishes to the laminar flow hood.
      
      
   4. Add the appropriate volume of antibiotic to the agar bottle before pouring.
      
      
   5. Pour the agar evenly to no higher than the raised line. NOTE: place the lid resting on the edge of the dish so as to catch any drops that fall from the bottle.
      
      
   6. Leave the agar plates to set (~15-30 minutes)
      
      
6. Heat shock the bacteria by placing them in the 42ºC water bath for NO MORE THAN 45 SECONDS.
   
   
7. Immediately place the bacteria on ICE for 1 minute.
   
   
8. Add 800µl LB and incubate at 37ºC for 1 hour.
   NOTE: Place Eppendorfs in a Petri dish before placing in incubator.
   
   
9. Plate the bacteria under sterile conditions (bunsen burner ON):
   
   
   1. Place a glass spreader in ethanol.
      
      
   2. Flame the spreader to burn off the ethanol and let it cool.
      
      
   3. Plate #1: spread 100µl of cell culture onto the plate
      
      
   4. Plate #2: spin down the remaining cells from step 3. Discard the supernatant and re-suspend the pellet in 100µl of fresh culture. Spread this onto a second plate as in previous step
      
      
10. Incubate at 37ºC overnight.




Transforming Competent Cells - NEB alpha-5 cells

Follow the protocol as above with the following changes:

If using the C2987 cells only use 50µl aliquots as supplied. Use 2µl of assembly product (1-5µl containing 1pg-100ng of plasmid DNA).

In step 6, heat shock the bacteria by placing them in the water bath for EXACTLY 30 seconds. Leave on ice for 5 minutes.

In step 8, add 950µl of SOC media and incubate as above.