PIRÁMIDE DE EDADES

Agarose gel loading buffer (5X): 50 mM Tris-HCl (pH 8.0), 50% (v /v ) glycerol, 1 m g /m l brom ophenol blue.

Alkaline gel buffer (lOx): 300 mM NaOH, 25 mM EDTA.

Alkaline phosphatase buffer: 100 mM Tris base, 100 mM NaCl, 50 mM MgCl^.

__________________________Chapter 2__________________________

A nnealing b u ffer (lOx): 100 mM Tris-HCl (pH 7.5), 500 mM NaCl, 100 mM MgCl,.

ATPase buffer: 50 mM triethanolamine-HCl (pH 7.5), 40 mM KCl, 1 mM MgCl^, 0.2 mM ATP, 1 mM DTT, lOOpg/ml BSA.

B inding buffer: 50 mM triethanolamine-HCl (pH 7.5), 40 mM KCl, 15 mM MgClj, 2 mM ATP, 1 mM DTT, 100 g g /m l BSA, 20 mM creatine phosphate and 2 U /m l creatine phosphokinase.

Blocking buffer: 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% (v /v ) calf serum , 3% (w /v ) BSA, 0.05% (v/v) Tween-20.

Coom assie b lu e staining solution: 40% (v/v) methanol, 10% (v /v ) glacial acetic acid, 250 p g /m l Coomassie brilliant blue R-250.

D estaining solution: 40% (v/v) methanol, 10% (v /v ) glacial acetic acid. D eveloping solution: 300 mM Na^CO^, 0.05% (v/v) formaldehyde.

Ferm enter broth: 32 g/1 bacto-tryptone, 20 g/1 bacto-yeast extract, 5 g/1 NaCl, 10 g/1 K2HPO4 and 1.85 g/1 KHjPO^, 10 mg/1 thiamine, 10 m g/1 biotin, 50 m g/1 thym ine, 100 m g/1 carbenicillin, 25 mg/1 chloramphenicol.

Form am ide loading buffer: 80% (v/v) deionized formamide, 89 mM Tris base, 89 mM boric acid, 2 mM EDTA, 1 m g /m l brom ophenol blue, 1 m g /m l xylene cyanol.

G el soaking buffer: 10 mM Tris-HCl (pH 8.0), 10% (v /v ) glycerol, 50 mM EDTA.

HI buffer: 30 mM HEPES-NaOH (pH 7.8), 1 mM DTT, 0.25 mM EDTA, 0.25% (w /v ) inositol, 0.01% (v /v ) Nonidet P-40.

hRP-A d ilu tio n buffer: 50 mM KOAc (pH 7.5).

H ypotonic lysis buffer: 20 mM Tris-HCl (pH 8.0), 10 mM NaCl, 5 mM NaF, 1 mM DTT, 0.019 T IU /m l aprotinin, 62.5 p g /m l PMSF.

Insect cell m edium : Grace's m edium supplemented w ith 3.3 g/1 TC-yeastolate, 3.3 g/1 TC-lactalbumin, 10% (v/v) heat inactivated foetal calf serum and 1% (v /v ) antibiotic/antim y cotic solution.

Chapter 2

Lurîa b ro th agar: 10 g/1 bacto-tryptone, 5 g/1 bacto-yeast extract, 5 g/1 NaCl, 15 g/1 agar.

Luria broth: 10 g/1 bacto-tryptone, 5 g/1 bacto-yeast extract, 5 g/1 NaCl.

Lysis b u ffer A: 100 mM Tris-HCl (pH 8.0), 2 mM EDTA, 0.5 mM DTT, 5% (v /v ) glycerol.

M inim al m edium agar (M9): 6 g/1 Na^HPO^ 7 H^O, 3 g/1 KH^PO^, 1 g/1 NH^Cl, 0.5 g/1 NaCl, 4 g/1 glucose, 15 g/1 agar.

M N d ilu tio n buffer: 20 mM Tris-HCl (pH 8.8), 25 mM CaCl^.

NEB bu ffer 4: 20 mM Tris-acetate (pH 7.9), 50 mM KOAc, 10 mM Mg(OAc)y 1 mM DTT.

P buffer: 50 mM potassium phosphate (pH 6.8), 10% (v /v ) glycerol, 0.5 mM DTT.

PlOO buffer: 100 mM potassium phosphate (pH 6.8), 100 mM KCl, 10% (v /v ) glycerol, 0.5 mM DTT.

Pairing b u ffer A: 50 mM triethanolamine-HCl (pH 7.5), 40 mM KCl, 2 mM MgCl,, 2 mM ATP, 1 mM DTT, 100 |ig /m l BSA.

Pairing b u ffer B: 50 mM triethanolamine-HCl (pH 7.5), 40 mM KOAc, 0.5 mM Mg(OAc)^, 2 mM ATP, 1 mM DTT, 100 p g /m l BSA.

Pairing b u ffer C: 50 mM triethanolamine-HCl (pH 7.5), 0.5 mM Mg(OAc)2, 2 mM ATP, 1 mM DTT, 100 p g /m l BSA.

PBS: 140 mM NaCl, 3.4 mM KCl, 10 mM Na^HPO,, 18 mM NaH^PO,.

Protein sam ple bu ffer (4X): 125 mM Tris-HCl (pH 6.8), 5% (v /v ) glycerol, 6 m g /m l SDS, 10% (v /v ) P-mercaptoethanol, 2 m g /m l brom ophenol blue.

Protein storage buffer: 20 mM Tris-acetate (pH 8.0), 200 mM KOAc, 10% (v /v ) glycerol, 1 mM EDTA, 0.5 mM DTT.

R buffer: 20 mM Tris-HCl (pH 8.0), 10% (v/v) glycerol, 1 mM EDTA, 1 mM DTT.

SDS gel b u ffer L: 375 mM Tris-HCl (pH 8.8), 1 m g /m l SDS. SDS gel b u ffer U: 125 mM Tris-HCl (pH 6.8), 1 m g /m l SDS.

Chapter 2

SDS gel ru n n in g buffer: 25 mM Tris base, 190 mM glycine, 1 m g /m l SDS. Sperm idine buffer: 20 mM Tris-acetate (pH 7.5), 7 mM sperm idine-N aOH (pH 7.5), 0.1 mM DTT.

Stop b u ffer A (5x): 100 mM Tris-HCl (pH 7.5), 200 mM EDTA, 25 m g /m l SDS, 10 m g /m l proteinase K

Stop b u ffer B (5x): 100 mM Tris-HCl (pH 7.5), 100 mM MgCl^, 25 m g /m l SDS, 10 m g /m l proteinase K, 2.5 p g /m l ethidium bromide.

TAE: 40 mM Tris base, 1.1% (v/v) glacial acetic acid, 1 mM EDTA. TBE: 89 mM Tris base, 89 mM boric acid, 2 mM EDTA.

TBST: 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.05% (v /v ) Tween-20. TE: 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA.

TNE: 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA. W estern b lot transfer buffer: 20 mM sodium phosphate (pH 6.7).

2.4 Bacterial strains

E. coli strain FB810 (Benson et al., 1994) is a recA derivative of BL21 (DE3) (Benson et al, 1994). Hum an Rad51 and hRP-A were expressed in FB810 by introducing plasm ids pFB530 and plld-tR P-A , respectively (sections 2.5). E. coli JM109 (Yanisch-Perron et al, 1985) was used for the production of RFI plasm id DNA (section 2.17), single-stranded phagem id DNA and M13 K 0 7 helper- phage (section 2.19). E. coli cells were grown in Luria broth (LB) or on LB agar plates unless stated otherwise. Cells were stored at -70°C in LB containing 30% glycerol (v/v).

2.5 Plasm ids

Plasm id D escription References

pFB530 £. coli expression vector p E T lld (Novagen) harbouring the hum an RAD 51 gene.

Studier et a l, 1990 Benson et a l, 1994

Chapter 2

pFB540 Baculovirus transfer vector pAcSC2 (Pharmingen) with the Ncol-BatnHL fragment of pFB530 (hum an RAD51 coding sequence) inserted at the Ncoï- Bglll sites.

F.E. Benson, unpublished

pLysS Expression of T7 lysozyme from the 03.8 prom oter in E. coli (Novagen).

Studier, 1991

p lld -tR P -A E. coli expression vector p E T lld (Novagen) harbouring the three subunits of hum an RP-A (hRP-A) under the control of a single OlO promoter.

Henricksen et al., 1994

pDEA-7Z f(+) Phagemid constructed by replacing the Scal-Bsal fragment of pCEM-7Z f(+) (Promega) with the Scal-Bsal fragment of pBR322.

Shah et ah, 1994a

pPB4.3 f(+) Derivative of pDEA-7Z f(+) generated by replacing the BamHl-HindlU fragment of pDEA-7Z f(+) w ith a 1276 bp fragment of X. laevis DNA {BamHl- Hindlll PCR fragment harbouring the cyclin E gene; gift from P. Descombes).

this work

pPB284 Phagemid constructed by inserting the 284 bp EcoRI fragment of pFB530 at the EcoRI site of the pBK-CMV vector (Stratagene).

this work

Plasm ids were m aintained in E. coli by adding the following antibiotics to the grow th medium: pFB530, plld-tR P-A , pDEA-7Z, pPB4.3 (100 p g /m l carbenicillin); pLysS (25 |ig /m l chloramphenicol); pBK-CMV, pPB284 (50 p g /m l kanamycin)