M13mp18 Phage Transfection
If starting from M13mp18 Single-Stranded DNA (N4040):
- 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.
- Fill-in reaction to form duplex. Add:
2 μL 10 mM each dNTPs
1 μL DNA polymerase (e.g. Klenow fragment)
- Room temperature incubation, 30 minutes.
- Heat kill at 65˚C.
- Store at –20˚C or transfect directly. Use 1 uL per 50 μL aliquot of competent cells.
- Transfections should be done into male bacterial strains, such as ER2738 or JM101.
- Add 0.5 μg of M13mp18 RFI DNA to 200 µl competent cells.
- Incubation for 30 minutes on ice
- Heat shock at 42°C for 2 minutes.
- Outgrowth step to let the cells grow before plating. It should be at least 30 to 45 minutes at 30°C.
- 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.
- Spill total sample on an LB plate.
- Incubation overnight at 37°C.
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.