M13 TRANSFORMATION OF BACTERIA
Preparation of Transformation Competent DNA
  1. Dilute an overnight bacterial culture 1/80 in L broth. You will need 2.5 ml of this culture per transformation plate, plus a few ml extra to determine the OD a few times during growth. When the A600 has reached 0.3 - 0.4, centrifuge the culture at 2500 rpm, for 20 minutes at 4°C. It usually takes 1 - 1.5 hours to reach this concentration. Discard the supernatant. Use a serological pipet to resuspend the bacterial pellet gently in 1/2 of the growth volume of cold 50 mM CaCl2. Keep on ice for 20 minutes. Melt top agar in a microwave and keep at 45°C until needed.
  2. Add 10 ml of L broth to the few bacteria remaining in the original flask and continue to grow to obtain an exponentially growing bacterial culture needed later in the protocol.
  3. Centrifuge the CaCl2 resuspended bacteria at 2500 rpm for 20 minutes at 4° C. Discard the supernatant.
  4. Resuspend the bacteria VERY GENTLY in 1/10 of the original growth volume with cold 50mM CaCl2. It is very important that you do this gently. Do not blast the cells! These CaCl2-treated bacteria are very fragile and the cells will lyse if they are vortexed or vigorously pipeted. The rougher you are at this point, the lower your transformation efficiency will be.
  5. For each transformation plate , aliquot 250 µl of this bacterial suspension to a 0.5 ml microcentrifuge tubes keep on ice.
Amount of DNA Needed per Petri Dish
Positive control: Use 0.5 ng of uncut M13 RF DNA.
Experimental ligation: Use 5 ng for overhanging-end ligated DNA, or 12 - 25 ng for blunt-end ligated DNA.
Transformation
  1. Add the appropriate amount of DNA to the bacteria while gently mixing with a pipet tip. Incubate on ice for 40 minutes.
  2. Heat shock these bacteria for 2 minutes at 42°C (37°C also works well).
  3. Add these bacteria to test tubes containing 0.2 ml of the freshly growing E. coli started earlier, 10 µl of 100 mM IPTG, and 25 µl of 4% Xgal. Add 3 ml of 45°C top agar and plate on a Petri dish containing LB agar.
  4. Start a 2 ml overnight culture of JM103 for expansion of the clones the next day.
  5. Determine the transformation efficiency the next morning by dividing the number of transformants per microgram of vector DNA. Good transformation efficiencies for the control plate are 1000 colonies per ng. Efficiencies for recombinant M13 will be lower and decrease with the size of the insert. Overhanging-end ligations are more efficient than blunt-end ligations. Pull colorless colonies from the plate by encircling them with the fine end of a Pasteur pipet and transfer each to 1.5 ml L broth with 20 µl of the saturated overnight bacterial culture. Incubate while shaking for 6 hours at 37°C. Centrifuge the bacteria at 2500 rpm for 12 minutes and save the supernatants. These contain the ssDNA M13 bacteriophage for DNA isolation.
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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. M13 Transformation of Bacteria. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/m13tx.html). 1997. Oklahoma City. Revision Date: October 2, 1997."