Except where stated below, all chemicals were purchased from BDH Chemicals Ltd., Poole, Dorset, and were of analytical reagent grade (AnalaR) wherever possible. Amersham International, Bucks, England
[>.32p] ¿ATP (50% ethanol), [ a - ^ S ] thio-dATP, N a*^Iodine. Protein molecular weight size markers [a-^2P] dATP (aqueous), [14C] methylated protein mixture. Anderman & Co., East Molesley, Surrey, England.
Nitrocellulose sheets, nitrocellulose filter discs, manufactured by Schleicher & Schull. Bethesda Research Laboratories (BRL/GIBCO), Paisley, Scotland
The following chemicals were ’ultra pure’ grade where possible. Urea, caesium chloride, agarose, ammonium persulphate, formamide, isopropyl thio-fi-galactoside (IPTG), phenol, tetramethylethylenediamine (TEMED), Lambda DNA, new bom calf serum.
Biorad Laboratories, Caxton Way, Watford, Herts Acrylamide, methylene-bis acrylamide.
Difco Laboratories, West Molesley, Surrey, England. Tryptone, yeast extract, agar.
ICN Biomedicals Ltd, High Wycombe, Bucks Gelbond
May and Baker Ltd. Glycerol, diethyl ether. Biocell Ltd, Cardiff, Wales Goat anti-rabbit labelled with gold.
Pharmacia (LKB) Ltd, Milton Keynes, England. Sephadex G25 columns (PD-10), Sephadex G50 Protein A.
Sigma Chemical Company (UK) Ltd., Poole, Dorset,England
Ethidium bromide, ampicillin, chloramphenicol, tetracycline, bromophenol Blue, xylene cyanol, orange G, ficoll, adenosine 5 * -triphosphate (ATP), DL-dithiothreitol (DTT), 5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside (BCIG/X-gal), isoamyl alcohol , dimethylformamide (DMF), polyethylene glycol (PEG; Mr 6000), sodium deoxycholate, polyvinylpyrrolidone, bovine serum albumin (BSA pentax fraction V), human IgG, salmon sperm DNA, thiamine, Triton X-100, Coomassie Brilliant Blue R250, polyoxyethylene 20 cetyl ether (Brij 58), nitrophenyl phosphate.
2.1.2 Enzymes
Boehringer Mannheim (BCL), Lewes, Sussex, U.K.
Calf intestinal alkaline phosphatase, T4 DNA polymerase, Klenow DNA polymerase I, Proteinase K, RNase T l, DNasel.
Bethesda Research Laboratories (BRL/GIBCO), Paisley, U.K. All restriction endonucleases, T4 polynucleotide ligase, T4 polynucleotide kinase. Sigma Chemical Co.Ltd.,Poole,U.K.
Lysozyme (Grade I), Lysostaphin 2.1.3 Media
All media listed below were prepared in double distilled, d e -ionised water and autoclaved at 15 p.s.i. (121°C) for 15 min unless otherwise stated.
2.1.3.1. Routine Growth Media
L-broth. This medium was routinely used for the cultivation o f E.coli during the protein A project.
The pH was adjusted to 7.4 using 1M NaOH. For so lid m ed ia (L-agar) 2%
(w/v/purified agar was added).
2xYT B roth: This media was routinely used for the cultivation o f E. coli during the alkaline phosphatase project and for the growth of phage containing E.coli JM101. Bacto-tryptone Yeast extract NaCl 10.0 5.0 5.0 29
Bacto-tryptone Bacto-yeast extract NaCl g l 1 16.0 10.0 10.0
The pH was adjusted to 7.4 using 1 M NaOH. For solid media (2xYT agar), 2% (w/v) purified agar was added.
Low phosphate broth (LP)
g l'1 Tris base 14.54 NaCl 4.68 KQ 1.49 NH4CI 1.07 Na2S04 0.97 MgCl2 0.203 CaCl2 0.0294 ZnCl2 0.0003
The pH was adjusted to 7.5 with 1M NaOH. For solid media LPB-agar, 1.5% (w/v) purified agar was added prior to autoclaving. Before use, the following supplements were added:
Final Concentration
Bactopeptone 5% autoclaved separately Casamino acids 0.05% autoclaved separately Thiamine 20pg/ml (filter sterilised) Glucose 0.2% (filter sterilised) Ampicillin 50pg/ml (filter sterilised)
H-Top Agar: This medium was used for the soft agar overlays in the cultivation of M13 phage plaques.
NaCl Agar
g l 1
8.0 8.0
The pH was adjusted to 7.4 with 1 M NaOH.
M9 Salts (lOx stock): This defined minimal medium was used for the cultivation of E. coli JM83 [SpA] g l 1 Na2HP04 300 k h2p o4 150 NaCl 25 n h4c i 150
The pH was adjusted to 7.4 with 10 M NaOH, and the mixture autoclaved. A volume o f 20 ml of this lOx stock was added to 180 ml molten (50°C) agar/water 2% (w/v) prior to the addition of the following constituents:
1 ml 100 mM MgS04 (autoclaved separately) 1 ml 10 mM C aC ^ (autoclaved separately) 2 ml 20% (w/v) Glucose (filter sterilised) 2.1.3.2. Antibiotics
Antibiotics were incorporated at appropriate concentrations in both solid and liquid media for the selection of resistant bacterial clones.
Ampicillin
Stock solution: 10 mg m l'1 of the sodium salt of ampicillin in distilled water. This was sterilised by filtration (0.22 pm Millipore disposable filter) and stored at -20°C. Working concentration: 50pg m l'1.
Chloramphenicol
Stock solution: 5 mg m l'1 in 100% ethanol (v/v). This was stored at -20°C. Working concentration for selection o f resistant bacteria: 30 pg m l'1, for amplification o f plasmids: 170pg m l'1.
Tetracycline
Stock solution 5 mg m l '1 o f tetracycline hydrochloride in ethanol/water (50% v/v). This was stored at -20°C in the dark.
Working concentration: ^ .S p g m r1. 2.1.4 Buffers and Solutions
All solutions listed below were prepared in deionised, double distilled water and autoclaved at 15p.s.i.(121°C) for 15 min unless otherwise stated.
TE Buffer (lx): Phosphate Buffered Saline Tris-HCl 10 mM (PBS): Na2EDTA 0.1 mM Na2HPQ4 8 mM pH 8.0 KH2P 04 1.5 mM NaCl 137 mM TES Buffer (lx): KC1 2.7 mM Tris-HCl 10 mM pH 7.4 Na2EDTA 0.1 mM
NaCl 170 mM Tricine buffer (4x):
pH 8.0 gl’ 1
Tris base 38.8
SSC Buffer (20x): Tricine 17.8
NaCl 3.0 M Calcium Lactate 0.5 Na3Citrate 0.3 M
pH 7.0 Ligation buffer (lOx): Tris-HCl 0.3 M
TBE Buffer (lOx): NaCl 0.3 M
Tris base 0.9 M MgCl2 0.075 M Boric acid 0.9 M Spermidine 0.01 M Na2EDTA 0.03 M ATP (Nasali) 0.0025 M
pH 8.3 DTT 0.02 M Na2EDTA 0.002 M TM buffer: pH 7.5 :Stored at -20°C. Tris-HCl 0.1 M MgCl2 0.05 M pH 8.5
Kinase buffer (lOx): Tris-HCl 0.5 M MgCl2 0.1 M DTT 0.05 M pH 7.4 Stored at -20°C T4 polymerase buffer (lOx) Tris-acetate 0.33 M K acetate 0.66 M Mg acetate 0.10 M DTT 0.005 M BSA (pentax Frac V) pH 7.9 Stored at -20°C. 1 mg ml" * S.T.E.T buffer: Sucrose 8 % (w/v) Triton X-100 5 % (v/v) Na2EDTA 0.05 M Tris-HCl pH 8.0 0.05 M Brij/Doc Solution Brij 58 1% Sodium deoxy- cholate (DOC) 0.4% Tris 0.01 M Na2EDTA pH8.0 0.001 M
Birnboim lysis solution (to make up 1ml) Lysozyme 200 mg Tris-HCl (pH8.0) 25 mM EDTA (ph8.0) 10 mM Glucose 50 mM (this solution was prepared on day of use) and stored Birnboim alkaline SDS Solution
NaOH 0.2 N
SDS 1%
RbCI Transformation buffer A
Mops pH7.0 10 mM RbCI 10 mM (this was sterilised by filtration u sin g a 0.22 pm M illipore disposable filter) and stored at 4°C
RbCI Transformation buffer B
Mops pH6.5 100 mM C a d 2 50 mM RbCI 10 mM (this was sterilised by filtration u sin g a 0.22 pm M illip o re disposable filter) and stored at 4°C
Agarose gel (Ficoll) tracking dye Ficol 50% w/v Bromophenol 0.02% w/v Xylene cyanol 0.02% w/v Orange G 0.08% w/v Denhardts reagent (50x) Polyvinylpyrrolidone 10 BSA (Pentax Frac V) 10 Ficoll 10 Filter sterilised and stored at -20°C. Prehybridisation solution SSC Denhardts reagent SDS Denatured SS salmon sperm DNA 6x lOx 0.2% (w/v) lOOngmT1
The salmon sperm DNA was denatured by heating to 100°C for 5 minutes. 40% (w/v) Acrylamide stock *11 Acrylamide 380 Bis-acrylamide 20
OJx TBE gel mix: 40% (w/v) acrylamide 150 ml lOx TBE 50 ml Urea (ultra-pure) 460 g Made up to 1 1 with distilled water.
5.0x TBE gel mix: