Selection for retention of the F factor by growth on minimal media is essential for JM101 and TG1. One should always grow fresh overnights in 2xYT from a colony on a minimal plate.
For preparation of RF, I quote: "When growing a bulk culture of M13 infected TG1, 10 ml. of a 1/100 dilution of an overnight culture is grown with shaking at 37° for 4 h. [toothpick a plaque into 1.5 ml. of a 100-fold dilution of an O/N culture of TG1 in 2xYT broth; shake standing vertically in a rack at greater than 300 rpm until it is visibly turbid (about 2 - 3 hours). Pour this infected culture into a 50 ml. tube holding 10 ml. of a growing culture of TG1, set up in parallel with the infected culture. Shake for a further 4-5 hours.] After centrifuging for 10 min. at 2000 rev./min, the supernatant containing the phage particles can be stored at 4° overnight [ We have found that this sort of culture supernatant can be stored frozen at -20° following growth in 2xYT for at least two years without significant loss of plaque-forming-units] and used to infect a fresh culture of cells at A660 of ~ 0.2. This is grown with shaking at 37° to A660 ~ 0.6 before proceeding with the chosen DNA purification technique." We've had good results allowing two 500ml. cultures to grow to A600 ~ 0.5 before inoculating at 5 ml. per flask, allowing growth with vigorous shaking for another 4 hours, then adding chloramphenicol at 25 µg/ml for the final 30 min. of growth.
Plaques are generally obtained by transfection; with phage; DNA. Dilute an overnight 1/100 into 2xYT and grow for 1.5-2 h with shaking at 37° to A660 ~0.3. Pellet cells at 4° and resuspend in one-half volume cold 50 mM CaCl2. Keep on ice 15-30'. Spin again and resuspend in one-tenth of the original volume cold 50 mM CaCl2. These competent cells do not freeze well, give the best results between 1 and 12 hours after being made, and are not useful after 48 h. To transfect 200 µl of these cells are aliquoted out into 13x100mm glass test tubes on ice, and ligation reactions or DNA (1 µl of a 1/100 dilution of a standard single-stranded M13 miniprep works well) is added directly, folowed by a 25-30 min. incubation. During this time top agar (0.7% in 1xYT) should be melted (at least 3 ml/plate). After the incubation the cells are heat-shocked by placing the tubes in a 42° temp-block for 5 minutes. During the heat-shock 3 ml. top agar (50° C), 25 µl of the TG1 O/N, 25 µl BCIG ("X-Gal", 25 mg/ml in dimethylformamide) and 25 µl IPTG (25 mg/ml filter-sterilized in water) per plate are combined at 50° [of course, the indicator and inducer are only needed when screening for inserts]; 3 ml. of this mixture is added to each tube, and the tubes are quickly mixed and poured. Generally we use standard lambda plates, but plates of 1.2% agar in 1xYT may give slightly better growth.
Single-stranded template DNA is prepared from plaques toothpicked into 2 ml.
aliquots of a 1/100 dilution of a fresh overnight placed in 13 x100 mm glass tubes.
These (multiples of 24 are most convenient) are grown standing in a vertical rack,
shaking at greater than 300 rpm, for 4.5-6 h. at 37°. Transfer 1.5 ml. into a microfuge
tube and spin 5'. Carefully pour or pipet the supernatant into a fresh microfuge tube
containing 200 µl of 20% PEG;, 2.0 M NaCl (polyethylene glycol 8000 from Sigma; this
solution should be filtered), being very careful not to carry over any of the pellet. Mix,
and allow this to stand for at least 10 minutes. Spin 10 minutes in a microfuge.
Aspirate the supernatant, spin again for ~ 1 min, aspirate again. Resuspend in 100 µl
TE (be sure the pellet is suspended before adding phenol). The most rigorous
extraction procedure is to vortex well, spin for 5 min, and transfer the supernatant,
using 1) 50 µl chloroform 2) 50 µl buffer-saturated phenol 3) 50µl of a 1:1
chloroform:phenol mixture. The simplest procedure is to use only phenol, and many
choose to do only the first two extractions. Following extraction, the aqueous phase is
brought to 150 mM NaOAc and precipitated with 250 µl of ethanol (usually overnight
at -20°). The pellet is washed once with 95% ethanol and resuspended in 30-40 µl of
TE. This volume can be adjusted somewhat in response to the size of the PEG pellets.