cDNA Library Screening.

2.1.3.1 Preparation of radiolabelled probe.

cD N A fragm ents w ere g e n erate d by re s tric tio n en zym e dig estio n for 2 h at 37°C and e le c tro p h o re se d on a 0.8% S e a p la q u e L ow M e ltin g a g a ro se gel (EM C B io p r o d u c ts ) . Fragment bands were excised using a razor blade, loaded onto a gel p u rific a tio n sp in -c o lu m n (M illip o re ) and c e n tr if u g e d at 3 5 0 0 rp m in a m ic ro fu g e fo r 4 m in . A d e s a ltin g c o lu m n (Pharmacia) was prespun at 2500rpm for Im in in a m icrofuge. The D N A sam ple was lo ad ed on the p resp u n c o lu m n and centrifuged at 2500rpm for Im in. The volum e was adjusted to 3 0 0 |i l with TE (pH7.5) and the D N A precipitated with 40^1 of 3M NaAc (pH5.2) and 800pl of 100% ethanol. Follow ing 5min incubation on dry ice, the sample was centrifuged at 13000 rpm in a m icro fu g e, the p ellet was w ash ed w ith 800pd o f 80% ethanol and re-centrifuged at 13000 rpm for 2min. The ethanol was discarded and the DNA pellet dried in a speedvac (Savant) and resuspended in 2 0 p l TE (pH7.5). D N A co n cen tratio n was

m easured using spectro p h o to m etry and its p u rity ch eck ed on an agarose gel.

R adioactive labelling of the probe was p erfo rm ed using the m ultiprim e kit (Amersham). 25ng of cDNA frag m en t in a volume of lOpd of water were boiled for 3min and the solution then chilled on ice for 2min. lOpl o f 5x m ultiprim e buffer and 5 p i of primer/BSA mix were added on ice, followed by lOpl of [ a ^ ^ P J d C T P (A m ersham nbr PB 10475, specific activity 6000 Ci/m m ol, less than one w eek old) and 2 p l o f K lenow D N A po ly m erase (NEB). The m ixture was left o v e rn ig h t at ro o m te m p e ra tu re . A fter o v e rn ig h t in c u b a tio n , th e r e a c tio n w as stopped by addition of 2 p l of 500 mM EDTA (pH8.0) and the p r o b e p u r i f i e d fro m u n i n c o r p o r a t e d n u c l e o t i d e s o n a Chrom aSpin 400 column (Clonetech). The colum n was prespun at 2800rpm for 2min. 1.5pl o f boiled carrier D N A (lO m g/m l) were added to the probes and the m ix tu re lo ad e d on the column, taking care not to break to matrix surface. The colum n was centrifuged for 2m in at 2800rpm in a m icrofuge and the eluant put in a fresh tube. A 2 p i aliquot was mixed with 8ml of s c in tilla tio n flu id (P a c k h a rd 6 0 1 3 3 8 9 ) and c o u n te d in a B eckm an instrum ent. The specific activity o f the pro b e was calculated using the following equation:

Specific activity = (total cpm X volume X 20) cpm /pg of DNA. Specific activities of 10^ c p m / p g could typically be achieved using this method. The probe could be stored at -20°C for a few days prior to use.

2.1.3.2 Screening procedure

15ml o f XL-1 B lu e c ells (S tra ta g e n e ) w e re g ro w n overnight at 37°C in L-Broth with lOmM M g S 0 4 . 1 and 4 p l of mouse whole embryo (days 11/12/13) cDNA library in XZ A P (kind gift of Dr P.Burbelo) were p u t at the bottom o f 15ml Falco n tubes, 2 0 0 p l o f bacterial culture were added and the tubes incubated for lOmin at 37°C. 10ml of L-Top agar (0.7% Agarose, lOmM M g S 0 4 ) were added to each Falcon tube and the m ix tu res p la te d on a 150mm dish w ith L -A g a r c o n ta in in g lOmM M g S 0 4 . The plates were left to dry at 37°C before being

in v e rte d and le ft o v e rn ig h t at 37°C. T he day after, the bacteriophage plaques were counted and the titre calculated.

A pproxim ately 10,000 to 20 ,0 0 0 p laq u es per dish were p la te d on eig h t 150m m d ish es as d e sc rib e d ab o v e. A fter o v e rn ig h t in cu b a tio n , the b a c te rio p h a g e w ere tra n s fe rre d to tw o 137m m n i t r o c e l l u lo s e m e m b r a n e c ir c le s ( S t r a ta g e n e 420107). The first m em brane was left on the plate for 3min and the second for 7 min. M em b ran es were m ark e d with a

syringe and pencil, soaked in IM NaOH for 30s, neutralised in

1.5M NaCl, 0.5M Tris-HCl (pHS.O) for 30s and rinsed in 2x SSC

for 30s (from 20x SSC: 0. M NaCitrate, 3M NaCl, pH adjusted to 7.0 with lOM NaOH). These three steps were carried out by placing membranes (phage side up) on large pieces o f W hatm an n u m b e r 1 p a p er so ak ed w ith the r e le v a n t so lu tio n s . T he m em branes were placed on a fresh piece of W hatm an paper to dry at ro o m te m p era tu re and th en b ak ed at 80°C (u n d er vacuum ) for 2h. Baked m em branes were preh y b rid ised in 200 ml of prehybridisation mix (lOx Denhardts, 6x SSC, 0.1% SDS) for 5h at 65°C. The radiolabelled probe was boiled for 5min and added to 50ml of hybridisation m ix (2x SSC, 0.1% SDS, Ix D enhardts) to 2.5x10^ cpm /m l and incubated with m em branes

at 65°C for 5h. The m em branes were then w ashed 3x with

200ml of 2x SSC, 0.1% SDS and 2x with 200ml of 0.2x SSC, 0.1%

SDS at room tem perature, then w rapped in Saran wrap and

exposed overnight to Kodak X-AR film. Radiactive spots present on both duplicates were m arked for further analysis.

In order to purify p o sitiv e b acterio p h ag e, p laq u es were subjected to two additional screens. First round positives were picked using the thick end o f a p asteu r pipette. T hese agar "plugs" were suspended in 500|xl o f phage buffer (lOOmM NaCl, lOmM M g S 0 4 , 50m M T ris-H C l (pH 7.5)) and the rem a in in g bacteria killed by addition o f a drop of chloroform . The agar plugs w ere left to soak fo r Ih at room te m p e ra tu re w ith interm ittent vortexing to resuspend the phage. The suspensions were kept at 4°C and titrated. For each putative positive clone, two 150mm dishes were prepared containing approxim ately 50 and 450 plaques. The plaques were transferred to nitrocellulose filters, denatured and screened as described above. A fter this

second round, individual p ositive plaques could be seen. To ensure purity of the b acterio p h ag e stocks, a third ro u n d of screening was carried out. A fter the third round, hom ogeneous bacteriophage stocks were obtained.

2.1.3.3 Recovery of plasmids from ÀZAP-based libraries

10ml cu ltu res o f XL-1 B lu e (S tra ta g e n e ) and SO L R (Stratagene) cells were grown in LB overnight in a 37°C shaker. The next morning, the cultures were diluted 1/100 in a volume of 50ml of LB. At O D 6 0 0 o f 0.2-0.5, the X L -Is centrifuged at 1500xg and resuspended to an O D 6 0 0 of 1.0 . 200p l of X L -1 Blue cells, 20 0 |il of phage stock to be rescued (containing less than 1 x 1 0 ^ phage particles) and I p l o f E xA ssist helper phage stock at 1x10^ pfu/pl (Stratagene) were added to a 50ml Falcon tube, mixed gently and left at 37°C for lOmin. M eanwhile, at O D 6 0 0 of 1.0, the SOLR cells were put in a 37°C incubator. 2ml of LB were added to the X L l/p h a g e mix and the tube was put in a 37°C shaker for 4h. The tube was heated at 70°C for 15min and centrifuged at 4000xg for 15min. The su p ern atan t co n tain in g the excised plasmid packaged into phage particles was decanted into a fresh tube and lOpl were added to 200|il of SOLR cells in a microfuge tube. After 15 min incubation at 37°C, 20 p i of this mix w ere plated on an L B -A g ar plate c o n tain in g lOOmg/ml ampicillin (Sigma). After overnight growth, colonies containing the rescued cDNAs in the vector pB luescript SK- were visible and glycerol stocks were made. The inserts were analysed by DNA sequencing.