Utilising the ArZap-cDNA cloning kit (Stratagene), and essentially following the supplier's recommended protocol, adult mice (8-10 week old cf) salivary gland random primed cDNA library was synthesised and screened as follows.
2.1 (!) Preparation of Salivaiy Qand RNA
Total RNA was isolated from mouse salivary glands by the LiCl-urea method (Aufifray and Rougen, 1980). After removing extraneous tissue, isolated salivary glands were homogenised using a polytron in ice-cold 3M LiCl containing 6M urea as the chaotropic agent. DNA in the lysate was sheared by sonicating the homogenate (output setting 25 for 20 seconds, 3 times on ice) prior to the salt-precipitation o f the released RNA by incubating the lysate overnight at 4°C. The precipitated RNA was centrifuged (7000 rpm, 10 min, 4°C, HB4 swing-out rotor), washed with half volume of cold 3M LiCl, 6M urea and re-centrifuged before dissolving in TE (lOmM Tris (pH 7.4), ImM EDTA, 0.5% SDS). The resulting solution was extracted once with phenol:chloroform:isoamyl alcohol (24:24:1) and ethanol precipitated overnight at - 20°C. The RNA was centrifuged (as above), washed with 75% ethanol, dissolved in sterile water and quantified spectrophotometrically (A260nm) before storing at -70°C at Ipg/pl.
2.1 (ii) Isolation of Poly (A)^ RNA
Poly (A)^ RNA was purified from the isolated total RNA using oligo (dT)-cellulose affinity column chromatography as described by Maniatis et al (1989). Working under RNAse-free conditions and at 4°C where possible, Img of total RNA was heat denatured (65°C, 2 min) and dissolved in an equal volume of sterile 2x loading buffer (4mM-Tris (pH 7.50, lOmM EDTA 0.2% SDS, IM NaCl). Prior to loading the
sample the oligo (dT)-cellulose affinity column (1cm x 0.25 cm^) was pre-equilibrated with sterile Ix loading buffer. The column was then loaded, under gravity flow with the denatured RNA sample and the eluant recirculated once. The column was then washed extensively with 5 column volumes of Ix loading buffer to elute all unbound RNA from the column. The bound poly (A)^ RNA was eluted from the column in 4 column volumes o f sterile elution buffer (lOmM Tris (pH 7.6), ImM EDTA, 0.05% SDS). The eluted Poly (A)^ RNA was ethanol precipitated, washed in 70% ethanol and redissolved in sterile water at Ipg/pl.
2.1 (iii) Libraiy Synthesis.
Using the reagents supplied and essentially keeping to the supplier's recommended protocol, the cDNA library was synthesised as follows. The first strand o f the cDNA was synthesised using M-MuLV reverse transcriptase by incubating 5.6 pg o f the salivary gland poly (A)^ RNA and 20ng of random hexanucleotide oligomers at 37°C for one hour. Second strand synthesis was performed at 16°C using DNA Polymerase I and RNase H. RNase H nicks the RNA in the RNA-DNA duplex to generate primers for DNA Polymerase I which synthesises the second strand to produce the cDNA duplex. The ends o f the resulting duplex were "polished" using the 3'-5' exonuclease and 5'-3' polymerase activity o f T4 DNA polymerase. EcoR I adaptors were ligated to the ends of the double strand cDNA molecules and the 5' cohesive ends o f the EcoR I adaptors were phosphorylated using T4 Polynucleotide kinase before ligation to the dephosphorylated cohesive ends of EcoR I digested XrZap vector arms.
The ligation product was packaged using Gigapack®!! packaging extract (Stratagene) and stored in SM buffer (lOOmM NaCl, 8mM MgS0 4.7H2 0, 2% Gelatin, 50mM Tris pH 7.5) containing 20pl chloroform at 4°C.
2.1 (iv) Libraiy Screening.
After assessing the titer o f the packaged library, approximately 10^ plaques were plated onto 20 15cm dishes (>50,000 pfu/plate) and screened with a c-ret mRNA
specific probe (Ikb Bam HI genomic sub-clone; Probe A Fig 3.2) as follows.
Bacterial host cells (E.coil DH5a) were prepared by inoculating a 50ml culture of LB containing 0.2% maltose and lOmM MgSO^ with a single colony and growing ovem iÿit at 30°C. The log-phase growing cells were spun down and resuspended in lOmM MgSO^ (O .D .;^= 0.5 of cell suspension).
For each plate, an aliquot equivalent to approximately 50,000 pfu from the packaged reaction was incubated at 37°C for 15 mins in 600pl of the freshly prepared D H 5a cells. The incubated sample was than mixed in 6.5ml of molten top agar (0.7% agarose in LB) at %40°C, poured onto the 15cm agar-plates (1.5% agar in LB) and left at room temperature to set before incubating inverted at 37°C for 6-8 hours. The plates were than cooled for 1-2 hours at 4°C before preceding with plaque transfer and hybridization.
2.1 (iv) (a) Plaque Lifts & H^bridizatioa
Phages from pre-cooled (4°C, 1-2 hours) library plates were carefully transferred, avoiding air bubbles, onto nitrocellulose membranes (Schleicher & Schuell) by placing the membrane on the plates for 1 min. After ensuring to mark the orientation of the filter on the plate, the membrane was carefully removed. The transferred phages were lysed by floating the membrane (phage side up) on 0.5MNaOH, 1.5MNaCl solution for 1 min prior to neutralisation by submerging in 1.5M NaCl, 0.5M Tris-HCl pH 8.0 for 5 mins and washing in 2xSSC for 1 min. The filters were air-dried on 3MM paper before covalent cross-linking the DNA onto the membrane by baking at 80°C for 2 hours. Prior to hybridization, the immobilised filters were re-immersed in 2xSSC and scrubbed in order to reduce any non-specific background.
Ifybridization: Filters were pre-hybridised in Hybaid roller bottles at 65°C for
1-2 hours in pre-hybridization solution (2xSSC, 0.1% SDS, lOx Denhardt's solution (i.e. 0.2% Ficoll, 0.2% BSA, 0.2% PVP), 50pg/pl denatured sheared salmon sperm DNA). After discarding the pre-hybridization solution, the filters were hybridized
o v em i^t at 65°C in hybridization solution (2xSSC, 0.1% SDS, lOx Denhardt's solution, 10% dextran sulfate (w/v), 50|ig/|il denatured sheared salmon sperm DNA) containing -10^ cpm/ml of ^^P-dCTP labelled nick translated probe. The probe was denatured by boiling for 2 mins prior to use.
Washes: The hybridized filters were washed in 2xSSC, 0.1% SDS for 5 mins at room temperature and than washed three times in 0.2xSSC, 0.1% SDS at 65°C for 20 mins each before o v em i^t exposure to X-ray film at -70°C.
Probes: In general double stranded DNA probes were radio-labelled with ^^P-dCTP by nick translation (Amersham Kit) (Kelly et al., 1970; Rigby et al., 1977). DNA to be labelled was gel-purified using the GeneClean® kit (BRL). This procedure relies on DNA adhering to glass at high salt concentrations and eluting at low salt concentrations. Basically the technique involved, size fiactionating restriction enzyme digested DNA by 1% TAEr-Agarose gel electrophoresis and isolating the required DNA band in minimal gel volume. The excised gel was dissolved in 3 volume (w/v) o f sodium iodide at 55°C for 10 mins prior to incubating with glass slurry (Ip l of slurry per 2pg o f DNA) on ice for 1 hour to bind DNA. The DNA-bound glass was microfuged (3000 rpm, 5 mins.) and washed once with cold Nal and twice with cold (-20°C) TNE/ethanol(l:l). The DNA was eluted fi-om the glass by re-suspending in TE (lOmM Tris (pH 7.4), ImM EDTA) and incubating the glass beads at 55°C for 20 mins. The mixture was centrifuged (15,000 rpm, 10 mins.) in order to separate and discard the glass beads firom the dissolved DNA solution.
lOOng of the isolated DNA was labelled in the presence of 20 |iM dGAT and lOpM (1 MBq (0.03 mCi) of ^^P-dCTP (3pi fi-om 370 MBq (10mCi)/ml; specific activity 110 TBq (3000 Ci)/mMol (Amersham)) with 0.5 units of DNA pol 1/ lOpg o f DNase I enzyme (Ip l o f enzyme mix) in lOpl total volume at 15°C for 90 mins. The labelling reaction was stopped with 5 pi phenol red (5% solution in TE) and the labelled probe was separated away fi-om the unincorporated nucleotides by size-exclusion chromatography on a small (7cm x 0.25cm^) Sephadex G50 column equilibrated with TE containing 0.1% SDS. The labelled DNA molecules elute as the first activity peak
and the smaller unincorporated label is retarded and co-elutes with the phenol red as the second activity peak. After monitoring the labelling efficiency of the probe by Cherenkov counting, the probe was denatured by boiling for 2 mins before adding to the hybridization mix (~10^ cpm/ml).
2.1 (iv) (b) Re-screening and Plaque Purificatioa
Positive plaques fi*om the primary screen were selected from the master plates by lining up the orientation marks on the film with those on the plates. The area around each positive plaques was picked, placed in 1ml of SM buffer (lOOmM NaCl, 8mM MgSO^.THzO, 2% Gelatin, 50mM Tris pH 7.5) and stored at 4°C. This stock was serially diluted, titered with host cells and re-screened as before. Clones remaining positive after the secondary screen were plaque-purified by another round of screening if necessary to unambiguously select a single positive recombinant. Plaque-purified recombinant phagemids were stored at 4°C in SOOpl of SM containing 20|li1 of
chloroform.
2.1 (iv) (c) In Vivo Excision of pBlueScript® SK(-) from X-Zap.
The ArZap vector consists of a modified À-phage genome in which the pBlueScript® SK(-) plasmid sequences have been inserted, flanked by the DNA synthesis initiation site at the 5' end and by the termination site of the "origin o f replication" of FI bacteriophage at the 3' end. The inserts in the recombinant phage are cloned in the EcoR I site of the multiple cloning site of pBlueScript® SK(-).
The ArZap in vivo excision strategy was utilised to directly sub-clone the insert from the recombinant phagemids selected from the library into the pBlueScript® SK(-) plasmid. This involves the co-infection of the recombinant Uni-Zap phage and a fl bacteriophage into XLl-Blue E.Coil host cells.
XLl-Blue host cells were prepared by inoculating a 50 ml culture of LB with a single colony and growing overnight at 37°C. Cells at log-phase (O.D.X6oo=hO) were spun
down and re-suspended in lOmM MgSO^ (O .D .;^= 1.0 of cell suspension). 200pl of the freshly prepared XLl-Blue cells were co-infected by incubating with 200pl o f the recombinant phage stock (> 10^ pfu) and Ipl of R408 helper phage (>10^ pfri) in a sterile 50ml conical flask at 37°C for 15 mins. 3mls of fresh LB was than added to the mixture and incubated further at 37°C for 2-2.5 hours. During this time period the co-infected cell use the helper phage and host proteins to replicate, circularise, package and secrete the recombinant pBlueScript® SK(-) phagemid into the culture medium. The host cells were than killed by incubating at 70°C for 20 mins and discarded by centrifugation (4,000 rpm, 5 mins.). The resulting supernatant containing the thermo-stable pBlueScript® SK(-) phagemid was stored at 4°C.
A 200|il aliquot o f fresh XLl-Blue host cells (O.D(X6oo)=LO) was used to infect with 50pl o f undiluted and lOpl of lOOx diluted phagemid stock respectively. The mixture was incubated at 37°C for 15 mins before plating out lOOpl aliquot on Ampicillin (50pg/ml) containing agar plates before incubating overnight at 37°C.
Colonies containing the recombinant pBlueScript® SK(-) plasmid were amplified and the DNA isolated by maxi preps (Maniatis et al., 1989). The insert DNA was isolated as described above and used to re-screen the library master filters.
All positive clones selected from the library screen were analyzed by restriction enzyme mapping. Southern blotting (section 2.6 (iii)) and sequencing before sub- cloning to produce a full length cDNA clone.