Home Protocols M13mp18 Phage Transfection

M13mp18 Phage Transfection

To obtain some M13mp18 phage from M13mp18 DNA, we recommend the following protocols:

If starting from M13mp18 Single-Stranded DNA (N4040): 
  1. Anneal primer to ssDNA:
    (13-x) μL MQ water
    x (2-5) μL seq template (~0.5 μg)
    2 μL 96 sequencing primer (2 pmol, about 20x template)
    2 μL NEBuffer 2.1

    Place in 95˚C water (microwaved in a beaker), allow to cool to 37˚C or room temperature.
     
  2. Fill-in reaction to form duplex. Add:
    2 μL 10 mM each dNTPs
    1 μL DNA polymerase (e.g. Klenow fragment) 

  3. Room temperature incubation, 30 minutes. 
  4. Heat kill at 65˚C. 
  5. Store at –20˚C or transfect directly. Use 1 uL per 50 μL aliquot of competent cells. 
  6. Transfections should be done into male bacterial strains, such as ER2738 or JM101.

If starting from M13mp18 RFI DNA (double-stranded, N4018):
  1. Add 0.5 μg of M13mp18 RFI DNA to 200 µl competent cells. 
  2. Incubation for 30 minutes on ice
  3. Heat shock at 42°C for 2 minutes.
  4. Outgrowth step to let the cells grow before plating. It should be at least 30 to 45 minutes at 30°C.
  5. Preparation of 4 mL Top Agar, adding of 200 µL of cell pre-culture to the transformation sample (to efficiently see a uniform bacterial lawn on the plate). Make sure the Top Agar is less than 55°C. Any higher temperature could kill the cells and dramatically reduce the transformation efficiency.
  6. Spill total sample on an LB plate.
  7. Incubation overnight at 37°C.
In parallel, a plate should also be prepared with only the pre-culture (without transformation sample), to efficiently compare coloring of the plates after incubation (blue color can appear very faintly).

Do several dilutions of the transformation samples before plating: 1:10, 1:100 and 1:1000 dilutions;

Add 10 μL of each dilution to the pre-culture before plating.

Also, check the recipe of the LB medium used. If it contains more than 5 g/L of NaCl, it is not appropriate for phage growth.

If a more detailed protocol is needed, please check our manual for the pH.D. Phage Display products, which contains all the protocols for phage transformation and growth, pages 8 to 9.