Team:Warwick/Protocols
Protocols
To ensure that our experiments went as smoothly and efficiently as possible, we followed tried and tested protocols.
Competent Cell Protocol
IMPORTANT: Pre-cool all the buffers and tubes that you use to 4ºC – This will take many hours in a fridge, over an hour on ice, the fastest is to use an ice/water slurry.
1. Take a single colony from an LB agar plate, inoculate in 5 ml of LB broth and incubate for overnight, 200 rpm at 370C.
2. Dilute 200 µL of culture in 100 ml of LB broth and incubate with shaking until the culture reaches an OD value of 0.30-0.35. You can make larger volumes if you like.
3. Keep the culture on in ice/water bath for 30 min, mixing every now and again to ensure even cooling. Make sure the culture never warms above 4ºC from this point on.
4.Transfer to pre-cooled centrifuge tubes and centrifuge at 3000 rpm at 40C for 10 min.
5. Remove the supernatant, then resuspend the bacteria in 10 ml of 100 mM CaCl2, mix well and incubate for 30 min on ice.
6. Centrifuge at 3000 rpm at 40C for 10 min. Discard the supernatant, then add 10 ml of 100 mM MgCl2, mix then incubate for 30 min on ice.
7. Centrifuge at 3000 rpm at 40C for 10 min and add 2 ml CaCl2 with 10% glycerol, resuspend bacteria.
8. Transfer 50 µL of culture into pre-cooled 1.5mL eppendorf tubes (on ice, or do this in the cold room). Snap freeze cells in liquid nitrogen and transfer to -80 freezer.
Ethanol Precipitation Protocol
Ethanol Precipitation of DNA Reagents Needed:
• 3 M sodium acetate pH 5.2 or 5 M ammonium acetate
• DNA
• 100% ethanol
Protocol
1. Measure the volume of the DNA sample.
2. Add 1/10 volume of sodium acetate, pH 5.2, (final concentration of 0.3 M) - These amounts assume that the DNA is in TE only; if DNA is in a solution containing salt, adjust salt accordingly to achieve the correct final concentration.
3. Mix well.
4. Add 2 to 2.5 volumes of cold 100% ethanol (calculated after salt addition).
5. Mix well.
6. Place on ice or at -20 degrees C for >20 minutes.
7. Spin a maximum speed in a microfuge 10-15 min.
8. Carefully decant supernatant.
9. Add 1 ml 70% ethanol. Mix. Spin briefly. Carefully decant supernatant.
10. Air dry or briefly vacuum dry pellet.
11. Resuspend pellet in the appropriate volume of TE or water.
Fluorescence microscopy
In order to visualise our cells and determine whether the modified zinc finger protein was binding to the surface of our E. coli cell, we also included a FLAG tag domain in the protein.
This allowed us to use anti-FLAG antibodies to conduct an immuno-fluorescence test to see the location of the protein in the cells.
