Preparing filter lifts o f the cDNA selection library
Hybond N-i- nylon membranes were cut to be the size as a 96-well m icrotitre plate and labelled using a pencil, with the plate number. M em branes were then placed on L-agar plates containing am picillin (lOOmg/ml). Plasm id clones were spotted onto the filters from the thawed microtitre tray glycerol stocks, using a 96-pin gridding tool. The agar plates were wrapped in Cling film and incubated overnight at 37°C such that the colonies had grown to 0.1- 0.2m m in diameter.
After incubation, filters were transferred from the L-agar plates onto a tray w ith W hatm an 3MM paper soaked in denaturing solution, were they were left for 5 minutes (DNA side up). Filters were then transferred onto a tray containing neutralising solution, where they were floated for another 5
minutes. Finally the filters were im m ersed in 2x SSC for 5minutes, bacterial debris was rem oved very gently and allowed to dry for 30 minutes. The filters w ere then baked at 80°C for 2 hours.
Southern blotting
A 0.8-1% agarose gel containing electrophoresed DNA was first placed in a shaking tray with denaturing solution for 30 minutes, rinsed in distilled w ater and then placed in a tray with neutralising solution for a further 30 minutes.
glass and dipping into the tray filled with lOx SSC. The gel was blotted onto H ybond N+ membrane, pre-soaked in 1 Ox SSC and covered with three layers of W hatm an paper 3MM, all of which were cut to fit the size of the agarose gel. A ir bubbles trapped in between the membrane and W hatm an paper 3M M layers, w ere rem oved and transfer of the DNA to the m em brane was achieved by capillary action, using a stack of paper towels and a weight on top of the assem bly to drive the vertical buffer flow. The gel was left to blot overnight. The m em brane was then removed, air dryed for 5-10 m inutes and baked in an
80°C oven for 2 hour
Prehybridisation o f DNA fixed onto filters
M embranes to be hybridised were placed into hybridisation bottles (Hybaid) with 50mls of hybridisation buffer and incubated for at least 2 hours in a tem perature equivalent to the hybridisation tem perature (57-65°C) in a rotating hybridisation oven (Hybaid).
Preparation o f Sephadex G50 spin columns
Rem oval of unincorporated nucleotides from the radiolabelling mix was done either by preparation and use of Sephadex G-50 spin colum ns, or by using the com m ercial Sephadex G-50 Nick-colum ns (Pharmacia).
The plunger from a 1ml syringe was rem oved and the end of the syringe was pluged with siliconised cotton wool. The syringe was then filled with Sephadex G50 suspension using a 1ml Gilson pipette avoiding any air bubbles. The syringe was then placed into a conical 5ml plastic tube and centrifuged at l,500rpm for 3 minutes. If necessary, the syringe was filled with Sephadex G50 again and re-centrifuged to a final level o f 1ml. The Sephadex G50 colum n was equilibrated by adding 200ml o f Ix TE and centrifuging for 1 m inute at l,500rpm . Columns were kept at 4°C until ready for use.
Preparation of ^^P-labelled probes
DNA used as probes were labelled by random prim ing (Feinberg and Vogelstein; 1984), using the Klenow fragm ent of DNA polym erase I to synthesise the second strand of DNA using denatured double-stranded tem plate DNA and random oligonucleotide primers. DNA was labelled with [a-^^P]dCTP, using the redi^nmo, DNA labelling system (Am ersham
Pharm acia Biotech). This system contains a dried, stable labelling mix of dATP, dGTP, dTTP, Klenow enzyme and random prim ers (9mers). In brief, approxim ately 25ng of DNA were diluted to a volum e of 45 pi in sterile distilled water, denatured by boiling for 5 minutes and added to the labelling mix, together with 2-4pl o f [a-^^P]dCTP. After incubation at 37°C for 30-45 minutes, unincorporated [a-^^P]dCTP and primers were rem oved by passing the labelling reaction through a Sephadex G-50 column. The labelling reaction volum e (50pl) was applied onto the filter matrix and eluted with 400pl IX TE. Percentage incorporation was estim ated by com paring the cpm retained on the G-50 Sephadex column, which represent unincorporated [a- ^^P]dCTP, with the cpm in the eluate. Successful incorporation was generally 60-80%. The DNA probe was denatured by boiling for 5 minutes before addition to the hybridisation solution and filters. H ybridisation buffer was reduced from 50mls to lOmls during radioactive labelling hybridisation.
Post-hybridisation washes and signal detection
The m em branes were prim arily w ashed at low stringency (2X SSC) for 5 m inutes at tem perature which could vary from room tem perature to 65°C and thereafter at increasingly higher stringency up to O.lx SSC and 0.1% SDS (57°C for hybridisation to different species, 65°C for hybridisation to same species). The m em branes were then briefly blotted on W hatm an 3M M paper to rem ove any excess liquid, wrapped in Cling film and exposed to X-ray film
at -70^C (rarely at room tem perature) in a light-proof cassette with intensifying screens. Exposures ranged from 4 hours to 4 days. All
autoradiography using and isotopes was carried out by exposure of X -ray film (Kodak biom ax MR), at room tem perature for ^^P and and with an intensifying screen at -70°C for ^^P.
R em oval o f radioactive probe from the filters
Radioactive probes were rem oved from the m em branes by boiling a large volum e of O.lx SSC, 1% SDS and place on the m em branes for 10-15 m inutes in a shaking tray. The above procedure was repeated three tim es, or until only a very low level of radioactivity could be detected using a hand-held G eiger counter. M embranes were then air-dried and stored at room
temperature.