LB agar

E.coli K12 TGI were grown to log phase (up to OD6oo=0.20-0.30) in Luria-Bertani (LB) broth, washed and ultimately concentrated 25 times in ice-cold 100 mM of CaCb. DNA was extracted from agarose gel after electrophoresis, added to 200 ml of competent cell and incubated at 0°C for 15 min. The cell-DNA complex was transferred to 42°C for exactly 90 s and was rapidly chilled in ice. Then 1000 ml LB-broth was added and the cells were incubated at 37°C for 60 min. 100 ml cells was spread on LB-agar with and without selective marker ampicillin (50 mg/ml), to obtain the number of transformants and viable cells respectively. Plates were incubated at 37°C for 18-24 h. 

E.coli recA y.luxCDABE strain were grown for 16-18 hours at 37°C in LB-broth in the presence of 20 pg/ ml of ampicillin. Immediately before the experiment the culture was diluted 1 20 by fresh culture medium and incubated until early log-phase. The grown biomass was mixed with AR solutions in final concentrations of ICfs, ICH n ICfs M, with used for their dilution with distilled water (control) and incubated for 60 minutes. The luminescence intensity of UV-irradiated E.coli recA lux and intact specimens were registered by plate bioluminometer LM OIT (Immimotech, Czech Rep.) in a real time. The number of viable cells was determined from the colony-forming units (CFU) on a surface of a LB-agar after the subsequent incubation within 24 hours at 37 °C. A quantitative estimation of an induction of the SOS-system calculated on formula... 

On solid medium, Azospirilhim and Escherichia coli strains were plated on LB agar with l%(w/v) pectin and after six days of incubation, the plates were overlayed with 2% (w/v) solution of hexadecyltrimetyl ammonium bromide (HTAB) (Plazinski and... 

Luria-Bertani (LB) liquid medium and LB agar plates containing ampicillin... 

To transform the E. coli cells, a 0.1 ml aliquot of competent cells was mixed with 1 jA plasmid DNA (1-10 ng). The mixture was left on ice for 10-30 min and then heated at 42 °C for 90 s. After adding 0.4 ml of LB, the cell suspension was incubated at 37 °C and then dilutions were plated on LB agar plates containing 50 pg ml of ampicUlin. Incubation was then overnight at 30 °C. For more details, consult the Stratagene manual (http //www.stratagene.eom/manuals/200133.pdf). 

Crystals from a frozen glycerol stock of E. coli BL21(DE3)/pPV2.85 were streaked onto LB agar plates with ampicillin (100 pg mL ) to obtain single colonies. 

Transform into E.coU with competency >10 c.f.u./pg DNA, Normally plated on 4 X 24-weU LB Agar Plates supplemented with Kanamycin... 

Transform competent coli (with a competency greater than 10 c.f.u./pig DNA) with 1 xl of the annealing reaction. Select for recombinants by plating on LB agar... 

Plate on 24-well format LB agar supplemented with antibiotic and, if appropriate, X-Gal and IPTG (dilute a 20% X-Gal, in dimethyl formamide, stock 1 1000, dilute the IPTG 500 mM stock 1 500 in warm agar before pouring). Plate 10 pi of cells, shake plates well to spread the cell suspension, and allow at least 10-15 min for the plates... 

After streaking the transformed cells on ampicillin-selective LB-agar plates, grow the cells overnight. 

One hundred microliters of the culture is spotted onto a square LB agar plate containing 50 pg/mL of kanamycin. Several spots and one streak are formed on the LB agar plate for each... 

Thirty microliters of the suspension is spread on one quarter of a square LB agar plate containing 50 pg/mL of kan-amycin and incubated at 37°C overnight. Thus four kinds of samples are on one plate and 24 plates are needed per a 96-sample set. In addition, glycerol stocks are prepared by blending 100 pL of the cell suspension with 50 pL of Cell Stock Buffer in a 96-well format. 

LB Agar Powder Growth Media, pH 7 (Mo Bio Laboratories) Store at room temperature. 

Pick up bacterial cells from the LB agar plate by wide side of teeth pick and rinse them into 1 ml of LBG medium in 96-deep-well plate covered with an AirPore Tape Sheet. 

LB agar add 1.5g Bacto-agar/100 mL LB broth. Sterilize by autoclaving... 

Plate the transformation mixture onto LB agar plates containing 100 pg/mL ampi-cillin and 1% glucose, and incubate overnight at 37°C... 

Add 10 mL warm SB (containing 20 pg/mL carbenicillin), remove 1- and 10-pL aliquots from this culture, and grow overnight on LB agar plates containing 50 pg/mL carbenicillin to titer the library size. Continue growth of the main culture for 1 h at 37°C... 

Self-ligate the DNA and transform into XLl-Blue by any standard method, since high efficiency is not crucial, and plate out on LB agar plates containing 50 pg/mL carbemcilhn The resultant clones can then be tested for Fab expression under IPTG induction. 

Spread the electroporation mix on a large petri dish containing LB agar and the appropriate antibiotic (tetracycline for fd-DOGl phages). Incubate overnight at 37 °C or 72 h at 23 °C. 

LB agar plates LB broth +16 g/L of bactoagar (Difco, Detroit, MI). Autoclave, allow to cool down to 45-50°C and pour into Petri dishes. 

LB agar plates + 40 pg/mL tetracycline + 100 pg/mL kanamycin. Antibiotics are added after autoclaving, just before pouring into Petri dishes. 

Transform 1.3 pL of the annealing reaction (diluted 1 5 with H20) into NovaBlue competent cells and plate the entire reaction onto LB agar plates containing 50 pg/ mL kanamycin. Incubate overnight at 37°C. 

Plate 50 pL onto prewarmed LB agar plates containing 50 pg/mL kanamycin, and grow overnight at 37°C. 

Transform, coli XLl-blue cells with the pQE-80LH6-BCCP-p53 plasmidDNA (or the plasmids encoding the individual mutants) using standard methods (18). Plate cells on LB agar plates supplemented with ampicillin at 100 pg/mL and incubate overnight at 37°C. 

Luria-Bertani (LB) agar, Mueller-Hinton (MH) agar or M9 agar (all from Difco Laboratories). The choice of the medium depends on the peptide used. If the peptide s activity is salt-sensitive, use of a low-salt minimal medium such as M9 is more appropriate. 

Fig. 4.4 Phenotype of 4-hydroxybutyrate dehydrogenase-positive (A) and lipase/esterase-positive (B) E. coli clones. A, 4-Hydroxy-butyrate dehydrogenase-positive clones are marked by arrows. Tetrazolium indicator plates [37] containing 4-hydroxybutyrate as test substrate were employed for the screening procedure [9], Positive clones were identified by formation of a deep-red formazan inside the colonies. B, Lipase/esterase activity of the clones was detected on LB agar [32] containing tributyrin as test substrate [14]. Zones of clearance around the colonies were indicative for lipase/ esterase activity.

Nonsense mutations and Tn5 insertions in secB are tolerated in haploid cells, indicating that secB is not an essential gene. However, secB null strains are sensitive to rich media and do not form isolated colonies on LB agar plates (Kumamoto and Beckwith, 1985). Induction of the heat-... 

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