Transfection of M13 DNA Protocol
This protocol includes transfection followed by phage titer steps, e.g. plaque assay, of media outgrowth. The Ph.D. Phage Display Manual also has a transfection protocol within Preparation of Electrocompetent Cells section. See also, General M13 Methods in the Ph.D. Manual. Either 50 or 100 µl frozen aliquots of homemade electrocompetent ER2738 E. coli are used. To use NEB Turbo Chemically Competent (NEB #C2984) or NEB Turbo Electrocompetent (NEB #C2986), please follow the product associated protocol with 45 min outgrowth period. Materials necessary for phage titer as well as phage replicative form (RF) DNA. Ph.D. Cloning System (NEB #E8101S) contains 1 µg/µl M13KE Phage RF DNA. Other M13 phage DNA products are M13mp18 RF I DNA (NEB #N4018S) and M13mp18 single-stranded DNA (NEB #N4040). Single-stranded DNA must be made partially double-stranded prior to transfection.
Materials
Day culture: ~10 mL fresh liquid F’ E. coli culture, e.g. ER2738, roughly log-phase
Molten (~48-52 ˚C) agarose top, 3 mL per sample
100 (or 50) µl aliquot competent cells
10-15 mL sterile culture tubes, e.g. VWR #47729-568, 17 mm x 100 mm tubes
SOC recovery medium, at room temperature, or ideally, 37 ˚C
LB/IPTG/XGal plates, pre-warmed 1 hr 37 ˚C
M13 RF DNA, freshly diluted in water to 1-5 ng/µl (NEB #E8101S contains M13KE at 1 µg/µl)
2 mm (or 1mm) electroporation cuvette, on ice, e.g. BTX #45-0134, #54-0135
Remove electrocompetent cells from freezer, and place on ice. Immediately, add 1-5 ng M13 RF DNA to the surface of the frozen cell pellet. Thaw on ice for approximately 5-10 minutes. Finger flick tube to confirm thaw and mix cells and DNA. Transfer cells to a chilled cuvette without introducing bubbles. Tap cuvette on hard surface to bring cells to the bottom and eliminate air bubbles.
Electroporate using the following conditions for Bio-Rad GenePulser electroporators: 2.5 kV, 200 Ω, and 25 μF (for 1 mm cuvette/50 µl aliquot:1-5 ng DNA, 2.1 kV, 100 Ω, and 25 μF)
Immediately add 1 mL of 37°C SOC to the cuvette, then transfer to culture tube. Shake vigorously (250 rpm) or rotate at 37 °C for 30-45 min.
Prepare 103 and 104 dilutions of the outgrowth. Transfer 10 μl outgrowth dilution to a sterile tube of 200 μl day culture cells and 3 mL agarose top. Briefly mix by vortexing a few seconds. Pour on to pre-warmed LB/IPTG/XGal plate. Gently, tilt and rotate plate to spread to agarose evenly. Incubate plates overnight at 37°C.
Media and Solutions
LB Medium:
10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl. Autoclave, store at room temperature.
Agarose Top
Per liter: 10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl, 7 g electrophoresis grade agarose. Dispense into 100-200 mL aliquots. Autoclave and microwave, as needed.
IPTG/Xgal Stock Solution
Mix 1.25 g IPTG (isopropyl-β-D-thiogalactoside) and 1 g Xgal (5-Bromo-4-chloro-3-indolyl-β-D-galactoside) in 25 ml DMF (dimethyl formamide). Solution can be stored at –20°C.
LB/IPTG/Xgal Plates
Per liter: liter LB medium, 15 g agar. Autoclave, cool to < 70°C, add 1 ml IPTG/Xgal Stock per liter and pour. Store plates at 4°C in the dark.
SOC Medium
Per liter: 20 g Bacto-Tryptone, 5 g yeast extract, 0.5 g NaCl. Dispense into 100 ml aliquots, autoclave and then store at room temperature. Prior to use, add 0.5 ml 2 M MgCl2 and 2 ml 1 M glucose (both sterile) per 100 ml. Then store at 4°C.
