I. Preparing E. coli Cell s for plating
A. Prepare a 50 ml liquid broth culture.
1. Prepare as for LB broth
.5 g tryptone
.25 g yeast extract
.25 g NaCl
50 ml 1 N NaOH
to 50 ml with dH2O
autoclave in 125 ml Erlenmeyer flask.
2.Broth must be 0.2% maltose and 10mM MgSO4. I keep a filter-sterilized 20% maltose stock and autoclaved 1M MgSO4 stock around; you can add .5 ml of each of these to a 50 ml broth culture either before or after autoclaving.
3. A. Inoculate with a single bacterial colony from a streak plate.
For new cDNA libraries (in lambda Zap) use XL1-Blu MRF’ cells.
For the cDNA library (in lambda gt11) use Y 1090 cells.
For the genomic library (in EMBL3) use KW251 cells.
C.Mix well, pour into a 50 ml conical bottom tube, and spin down 10 min at 1500 x g in the Beckman centrifuge.
D.Pour off medium and resuspend cells in 10 ml of sterile 10mM MgSO4.
E. Measure A600 of this suspension. An A600 of 2 indicates 1.5 x 109 cells/ml. The absorbance should be about 1; if it’s way over this, dilute and re-measure. Calculate the cells/ml accordingly. This suspension may be stored at 4° C for up to one week. Label the tube with the date, what strain of E. coli it is, the A600 reading, and how many ml to use for 2 x 108 cells.
A.Prepare LB plates and LB top agar (see Short Protocols p. 1-4.)
B.Plates are best used after at least three days of aging (unwrapped), so they"ll be somewhat dry. Pour the media as cool as possible for the same reason.
C.Melt top agar in microwave and clamp flask in water bath at 50° C. Place plates in 37° incubator to warm them.
D. In sterile 15 ml conical bottom tube, place enough of the E. coli suspension to provide 2 x 108 cells.
E.Add diluted phage to the tube. If you grow phage about 6 hours, you can probably get 10,000 plaques on a regular petri dish without them being quite confluent. If you want to be able to pick clean plaques the first time, probably no more than 1000 pfu per plate will work. In rescreening, the number of pfu per plaque may vary considerably. From big plugs, try about 5 ml of 10-2 dilution of a 500 ml SM picked plaque; for single plaques, use 2-10 ml of a 500 ml SM picked plaque. These dilutions should give 100 to 500 plaques when plated out.