Materials

2.1.1 Chemicals

All chemicals used in this project were supplied by BDH, Poole, Dorset, UK with the following exceptions.

35SdATP - Amersham, Little Chalfont, Bucks, UK

D eoxynucleoside triphosphates (dNTP) - P rom ega,S outham pton, Hampshire, UK

Phenol - Fisons, Loughborough, Leicestershire, UK RNAzol B - Cinna/Biotecx, Friendswood, Texas, USA

2.1.2 Plasmids and oligonucleotides

Vector pGEM-T was supplied by Promega

Vectors pBCVHCASS4, p G ID I, pKNIOO and pLNIO were kind gifts from Dr C.A. Kettleborough, MRC Collaborative Unit, Mill Hill, London.

All oligonucleotide primers for use in the polymerase chain reaction (PCR) were supplied by Genosys, Cambridge, Cambridgeshire, UK

2.1.3 Bacterial strains

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Escherichia coii of strain DH5a were supplied by Gibco, Paisley, UK

2.1.4 Eukaryotic cell lines

COS-7 cells were a gift from Mrs Alison Levy, MRC Collaborative Unit, Mill Hill, London,UK. They were originally derived from American Type Culture Collection, Ref No. CRL 1651.

The hybridoma lines LJ-1, AH-2, DA-3, UK-4 and RH-14 were all produced by Dr Sanjeev Menon and Dr Chelliah Ravirajan at the Centre for Rheumatology/Bloomsbury Rheumatology Unit, University College, London, UK. They were produced by fusion of PBL from patients with SLE with the mouse/human heteromyeloma line CB-F7. The characteristics of these patients are described in table 3.1 and the binding properties of the mAb LJ- 1, AH-2, DA-3, UK-4 and RH-14 in table 3.2 (see chapter 3).

2.1.5 Enzvmes

All restriction enzymes, DNA modifying enzymes and RNase inhibitor were supplied by Promega with the following exceptions :-

Sfil - New England Biolabs (NEB), Beverley, Massachusetts, USA Tag polymerase - USB/ Amersham, Little Chalfont, Bucks, UK Sequenase version 2.0 (DNA polymerase) - USB Amersham DNase I - Boehringer, Mannheim, Germany

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2.1.6 Bacterial growth media

Luria-Bertani (LB) medium was made according to the following recipe

for 11 medium

lOg Bacto-tryptone (Difco, Detroit, Michigan, USA) 5g Bacto-yeast extract (Difco)

lOg NaCI

Where it was necessary to add antibiotic to this medium the final concentrations used were lOOpg/ml ampicillin or 50|ig/ml chloramphenicol. Antibiotics were kept as stock solutions at -20°C in light-tight containers. Ampicillin was stored at a concentration of 50mg/ml in sterile water and chloramphenicol at a concentration of 34mg/ml in ethanol.

To make plates for growth of bacteria, 6g agar were added to 400ml of LB medium. The resulting "LB agar" was autoclaved, allowed to cool to hand- heat, then poured into 10cm petri dishes and allowed to set. Concentrations of antibiotic added to these plates were SOpg/ml ampicillin or 50pg/m l chloramphenicol.

2.1.7 Materials for gel electrophoresis

Agarose and low melting point agarose were supplied by Boehringer.

Polyacrylamide gels for sequencing were prepared using gel concentrate and diluent supplied by Scotlab, Coatbridge, Strathclyde, UK. The recipe used to prepare 50ml gel solution was:-

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38ml Diluent Solution

12ml Concentrate (containing 7M urea and acrylamide and bis-acrylamide in a ratio of 20:1)

2 2 0 |il ammonium persulphate solution (made freshly as 230mg in 1ml sterile water)

30|il N,N,N',N' tetramethylethylenediamine (TEMED)

2.1.8 Growth media for COS-7 cells

A) Medium for maintenance of cells in culture was made up as follows

500ml Dulbecco's Modified Eagle Medium (Gibco) 55ml Fetal Bovine Serum (Gibco)

6ml L-glutamine (58/mg/ml) (Gibco)

3ml of a solution containing 10000units/ml penicillin and lOm g/ml streptomycin (Gibco)

B) Medium for incubation of cells after electroporation was made up as follows

% 500ml Dulbecco's Modified Eagle Medium

25ml Ultra Low Ig Fetal Bovine Serum (Gibco) 6ml L-glutamine (as above)

3ml penicillin/streptomycin (as above)

2.1.9 Materials used in enzyme-linked immunosorbent assay (ELISA)

All IgG standards and goat anti-human IgG alkaline phosphatase conjugates were supplied by Sigma (Poole, Dorset, UK). Conjugates were supplied at a concentration of 3500 enzyme units per ml.

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Solutions of ssDNA and dsDNA were a kind gift from Dr C T . Ravirajan,

p nitrophenyl phosphate ( p NPP) substrate for development of a coloured phosphatase product was supplied by Sigma.

ELISA plates (Nunc Immunosorp) were supplied by Gibco.

The plate washer and plate reader were both supplied by Dynatech, St Albans, Hertfordshire, UK.

2.1.10 Buffers

The following buffers were used

a) lOxPCR Buffer - supplied by Promega 500m M KCI

lOOmM Tris MCI (pH 9.0 at 25°C) 1% Triton X-100

b) DNA ligase buffer - supplied by Promega, Southampton , UK 300mM Tris HCI

lOOmM MgCl2

lOOmM dithiothreitol (DTT)

5mM adenosine triphosphate (ATP)

c) Promega Buffer E - for restriction enzymes BamHI and Hindi!! 6mM Tris HCI (pH 7.5 at 25°C)

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1Q0mM NaCI 1mM DTT

d) New England Biolabs Buffer 2 - for restriction enzyme Sfil 1GmM Tris HCI (pH 7.9 at 25°C)

10mM MgCl2

50mM NaCI 1mM DTT

lOOpg/ml Bovine Serum Albumin (BSA)

e) Sequenase 5x reaction buffer (supplied by USB/Amersham) 200mM Tris HCI (pH 7.5 at 25°C)

lOOmM MgCl2

250mM NaCI

f) Tris EDTA (TE buffer) 1Q0mM Tris HCI 50nnM EDTA

g) Tris Borate EDTA (TBE buffer)

TBE buffer was made up as a stock solution of ten times working strength by dissolving components in sterile water as follows

108.9g/l Tris HCI 55.7g/l Boric acid 4.7g/l EDTA

The final concentrations after dilution to working strength were 45mM Tris- borate and 1mm EDTA.

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h) Tris Acetate EDTA (TAE buffer)

TAE buffer was made up as a stock solution of fifty times working strength by dissolving components in sterile water as follows

242g/l Tris HCI

57mI/I glacial acetic acid 39.2g/l EDTA

The final concentrations after dilution to working strength were 40mM Tris acetate and 1mM EDTA.

i) Phosphate buffered saline (PBS) pH 7.4

The following components were added to 101 of sterile water 800g NaCI

20g KCI

114.8g Na2HP0 4.2H2 0

20g K H2P O4

To make PBS / 0.1%Tween , 10ml "Tween 20" detergent was added to 101 PBS.

j) Bicarbonate buffer (pH 9.6)

To make 101, the following components were dissolved in sterile water Bg Na2C0 3

15.5g NaHCOa

k) Sample enzyme conjugate (SEC) buffer for ELISA lOOmMTris HCI

lOOmM NaCI 0.02% Tween (v/v)

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2g/l BSA

I) Loading Buffer (for agarose gel electrophoresis)

The following components were added to 8.45ml autoclaved water 25mg bromophenol blue

25mg xylene cyanole 1.5g Ficoll 400

2.1.11 Miscellaneous

Tissue culture flasks and dishes were supplied by Gibco.

The "Gene Puiser" electroporator, and disposable cuvettes for use with it were supplied by BioRad, Hemel Hempstead, Hertfordshire, UK.

C entriprep 30 concentrators were supplied by Amicon, Beverley, Massachusetts, USA.