Screening human adult testis and mouse spermatocyte cDNA libraries A human adult testis cDNA library in lambda ZAP was titred and screened

according to the conditions advised by Stratagene, the suppliers of lambda ZAP, using XL 1-Blue as the host strain. Another cDNA library which had been made from mouse spermatocyte in Uni-ZAP (Stratagene) was also screened according to the manufacturer’s instruction (nearly the same for both libraries). The titre of the human and mouse libraries were estimated at 1 x 10^ and 2.5 x 10^ p.f.u./ml, respectively.

11.2.8.1 Preparation of competent ceils

E.coli strain XL 1-Blue was streaked onto L agar plates supplemented with

100p,g/ml ampicillin and incubated, inverted, at 37°C overnight. A single colony was then isolated and used to inoculate lOmls of L broth supplemented with lOmM MgS0 4 and 2% maltose, and shaken at 250rpm overnight at 37°C.

1ml of this overnight culture was added to 50mls of pre-warmed L broth (containing the same supplements as before) and shaken at 37°C until the cells grew to an ODôoo of 0.5 (approx. 2.5 x 10^ cells/ml). The culture was then cooled on ice and centrifuged at 1500rpm in a Mistral 2000 bench centrifuge for 10 minutes. The

cells were resuspended in 15mls of ice-cold sterile lOmM MgS0 4 which was made

up using filtered double distilled water. The cells were usually used immediately, but could be stored at 4°C for 2 weeks with little loss in plating efficiency (so 5- 10% more aged cells were used).

11.2.8.2 Plating out the cDNA library

Approximately 3x10^ pfu from the human adult testis cDNA library and 2 x 10^ pfu from the mouse spermatocyte cDNA library were each plated as follows onto four 22 x 22cm megaplates containing NZY agar (pre-warmed to 37°C). An appropriate dilution of the cDNA library was added to 2mls of competent cells and incubated at 37°C for 15 minutes. 50-60mls L top agarose was melted and cooled to 45°C and 1ml of 20% maltose was added before the cells were added and the top agarose was poured on top of an NZY agar plates. After setting, the plates were incubated at 37°C inverted, overnight.

Filter lifts were taken from the plates by placing a wet Hybond membrane after soaking in 2x SSC or ddH20 on each plate (orientated with pin-pricks and ink) for 2 minutes, and then a second, duplicate, membrane for 4 minutes. These filters were placed (DNA side up) on Whatman 3MM paper soaked with denaturing solution for 2 minutes, and then submerged in IM Tris base for 2 minutes and neutralizing solution for 5 minutes, twice. Before the filters were air dried and then baked in an 80°C oven for 2 hours they were soaked in 2x SSC for 2 minutes. Ipl of the unlabelled probes were used to make a dot on top of the filters to control the hybridisation.

11.2.8.3 Pre-hybridisation and hybridisation of DNA fixed onto filters Pre-hybridisation buffer which was used for hybridisation was 5x SSPE or SSC, l-3xDenhardt’s, 0.1-0.5% SDS, 100-200 pg/ml denatured, sheared. Herring sperm DNA and 5M saturated tetra-pyrophosphate sodium salt in ddH20.

Membranes to be hybridised were placed into hybridisation bottles (Hybaid) with 50mls of hybridisation buffer and incubated for at least 2 hours in a temperature

equivalent to the hybridisation temperature (60-65°C) in a rotating hybridisation oven (Hybaid).

The radiolabelled DNA probe was then denatured by boiling for 5 minutes and then added to the hybridisation buffer after removing about 2/3 of the buffer. Hybridisation was usually carried out overnight at 63-65°C in a shaking water bath or rotating hybridisation oven.

11.2.8.4 Washes after hybridisation and signal detection

The membranes were primarily washed with 2X SSC and 0.1% SDS twice at room temperature and then at low stringency with the same washing buffer for 20- 40 minutes at 60-65°C temperature and thereafter at increasingly higher stringency up to 0.1 X SSC and 0.1% SDS at room temperature for 20-40 minutes. The washing

conditions were varied depending on the probe and checking radioactivity of filters by a Geiger. The membranes were then briefly blotted on Whatman 3MM paper to remove any excess liquid, wrapped in Cling film and exposed to X-ray film (Kodak biomax MR) at -70°C in a light-proof cassette with intensifying screens. Exposures ranged from 16 hours to 3 days.

11.2.8.5 Isolation of positive plaques and preparation of amplified phage stocks

Positive plaques, present in both first and duplicated filters, were isolated from the original cDNA library plates and stored in 0.5ml of SM buffer containing 20pl of chloroform. Plaque plugs as small as possible were isolated in order to have less irrelevant plaques in the secondary screening. The plaques were allowed to diffuse for 1-2 hours at room temperature and then stored at 4°C. Further screenings were carried out by plating dilutions of these primary plaques onto 90mm agar plates so that secondary plaques could be isolated.

An amplified stock was made from single positive plaques and also both human and mouse libraries by plating about 10^ pfu onto 90mm agar plates and growing at 37°C overnight. Each plate was overlaid with 5mis of SM buffer and left at 4°C for 2 hours with intermittent shaking. The SM buffer was harvested and a

further 1ml was added to the plate for 15 minutes; this was then combined with the first harvest. 0.1ml of chloroform was added to the amplified stock, vortexed and centrifuged at SOOOrpm in a Mistral 2000 bench centrifuge for 10 minutes. The supernatant was then recovered, one drop of chloroform was added and the stock stored at 4°C. Amplified stocks had titers ranging from lO^^-lO^^ pfu/ml.

II.2.8.6 Isolation of cDNA inserts

The lamdaZAP and Uni-ZAP XR vector has been designed to allow in vivo

excision and recircularisation of any cloned insert contained into the lambda vector to form a phagemid (pBluescript) containing the cloned insert. This system provides efficient excision of the pBluescript phagemid from the vector, while eliminating problems associated with helper phage coinfection. The ExAssist helper phage contains an amber mutation that prevents replication of the phage genome in a nonsuppressing E.coli strain such as SOLR cells. This allows only the excised phagemid to replicate in the host, removing the possibility of productive coinfection from the ExAssist helper phage. A liquid lysate was prepared by incubation of 10^ XLl-Blue cells, 10^ of ExAssist helper phage and 10^ pfu of the isolated phage. The phage were allowed to adhere to the cells at 37°C for 15 minutes without shaking, and then by adding 3ml of LB broth were grown at 37°C for about 3 hours, shaking at ~250rpm. During this time, both phages grow, recombination excision of the phagemid takes place and cells lyse. The culture was centrifuged for 15 minutes at 3000 X g to remove cellular debris and intact cells. The culture supernatant was

heated at 70°C for 15 minutes and centrifuged for 15 minutes at 3000 x g. A mixture of 200pl of SOLR cells (OD6oo=l) and lOOpl of the heated phagemid solution after incubation at 37°C for 15 minutes was plated onto two or three plates containing LB agar and 50pg /ml ampicilin. After overnight incubation at 37°C colonies containing the pBluescript double-stranded phagemid with cloned DNA insert were checked by PCR.