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The human, pig and chicken genomic cosmid and mouse genomic phage libraries were screened for the region o f interest using standard procedures o f cosmid and phage library screening (Sambrook 1989). The procedures involved in screening cosmid libraries have been described in the next few sections and the procedures for phage library screening follows. Firstly, the titre o f the libraries were determined and then an appropriate volume, representing the whole genome was plated onto hybond membranes laid on LB agar (see section 2.13.2) mega plates. Replica Hybond N+ membranes (22 b y 22cm) were made o f the master library plates and these were then probed for the region o f interest.

2.11.4.1 Titre of cosmid libraries

The titre of the libraries was determined in the following manner. 1 p i o f the library was added to 999pl o f LB broth (see section 2.13.2) and mixed well. This constitutes a 1000 fold dilution. A 1000 fold serial dilution o f this original stock was set up until lO’^ to 10*^ fold dilution was achieved. A range o f volumes such as 50pl and lOOpl o f the last 5 series o f dilution were plated directly on to 90mm LB agar (see section 2.13.2) plates containing the appropriate antibiotics (see section 2.11.1). The plates were allowed to rest at room temperature for a few minutes and incubated inverted ovemight in a 37°C oven. The plate with the least spread o f colonies was then selected and the number o f colonies present on the plate counted. If the concentration o f colonies were too high, to even estimate the number o f colonies present on the plate, then additional 1000 fold serial dilutions were performed. The titre o f the library, represented by colony forming units (cfu) per pi o f library was estimated using the following equation:

cfu/pl = total no. o f colonies in lOOOpl o f 10^ x 10^'^

where 10^ represents the 1000 fold dilution at which the number o f colonies were determined.

The titre o f the genomic cosmid libraries screened were estimated to be: human genomic cosmid library = 5.0 x 10^ cfu/pl, pig genomic cosmid library = 2.9 x 10^ cfu/pl, chicken

Methods and Materials genomic cosmid library = 5.3 x 10^cfu/)il. After determining the titre o f each library, an aliquot o f the library was plated to generate enough colonies to represent the whole genome.

2.11.4.2 Preparation of genomic cosmid libraries for hybridisation

The libraries were thawed at room temperature. The titre o f the library was determined as described above. An appropriate volume o f the library representing a whole genome was plated directly onto membranes laid on LB agar mega plates containing the appropriate antibiotics. A minimum o f 5 x 10^, 5.8 x 10^, 2 x 10^ colonies were plated to make the human, pig and chicken genomic cosmid master library, respectively. Standard procedure was used to make the replica membranes o f the libraries except the chicken cosmid library which was prepared as specified by the manufacturers (Clontech Laboratories, USA). The protocols discussed in section 2.7.2 and 2.7.3 were followed for the prehybridising, hybridising, and washing o f the membranes to remove un- specifically bound probes, respectively. Membranes were hybridised in Denhardt’s reagent (see section 2.7.2) and the probes were labelled using the Ready-To-Go a-^^P- dCTP labelling kit and prepared as described in section 2.7.1.1. Positive colonies were identified, isolated and subjected to secondary and tertiary screening as described below.

Replicated positive cosmid colonies were identified from the X-ray autoradiograph o f the replica membranes. The position of the positive colonies on the master library plate was determined by aligning the X-ray autoradiograph with the master library plate, using the asymmetric orientation marks placed on the membranes. If a single isolated colony could not be picked, then the surrounding colonies were also picked and resuspended in 500p.l LB broth solution. The cells were then grown in a 37°C shaking incubator for about 1 hour. 10-20^11 o f this culture was then diluted in 500p,l LB broth. 50p.l, 100p.l and 300jil aliquot o f this dilution was plated onto 137mm round membranes placed on midi LB agar plates, containing the appropriate antibiotics. The cells were spread using a glass spreader and the plates were incubated inverted ovemight at 37°C. One plate with an even spread o f isolated colonies was selected to represent each positive colony picked initially and replica membranes were made. The replica membranes were probed, as performed previously for the primary screening. From the secondary screen X-ray autoradiograph, the positives were identified, isolated and subjected to a tertiary screen.

Methods and Materials For the tertiary screen, the positive colonies were spread on 82mm membranes laid on 90mm LB agar plates containing the appropriate antibiotics. By the third screening, an individual colony should have been isolated, such that 90% o f the spread o f colonies on the agar plate must be the same positive colony, if not then a fourth screening, was performed. Glycerol stocks were made o f all the positive colonies isolated from the various stages o f screening, by adding an equal volume o f 50% glycerol solution (autoclaved) to an aliquot o f the positive colony LB broth culture and stored at -80°C.

2.11.4.3 Titre of phage library

The titre o f the phage library was determined as follows. A serial dilution o f Ip l o f the phage library was set up in 999|Li1 SM buffer [0.58% (w/v) NaCl, 0.2% (w/v) M gS0 4.7H2 0, 0.05M Tris-HCl (pH7.5), 0.01% (w/v) gelatin and sterilised b y autoclaving] as discussed in section 2.11.4.1. A range o f aliquots such as 50|xl and lOOpl o f the last few dilutions were added to 1ml M RA -X Ll-Blue (P2) cells (see section 2.11.4.4) and incubated for 15 minutes in a 37°C oven. The cells absorb the phage particles at this stage. Once incubation is complete, 3mls o f top agar (see section 2.13.2) was added to the cells and the mixture plated directly onto 90mm LB agar plates. The plates were allowed to set for 10 minutes at room temperature and then incubated inverted ovemight at 37°C. The titre o f the library was calculated as described for the cosmid libraries. The titre o f the library was determined to be greater than 1.4 x 10^^ pfu/pl.

2.11.4.4 Preparation of competent XLl-Blue MRA (P2) cells

XL 1-Blue MRA cells were freshly prepared before every application. LB agar plates were streaked with XL 1-blue MRA cells to obtain single isolated colonies. The plates were inverted and incubated ovemight in a 37°C oven. An ovemight culture was set up in 10ml LB broth inoculated with a single colony o f cells and maltose was added to a final concentration o f 0.2% (w/v) [made with SDW and filter sterilised using 0.2jiM syringe filter (25mm surfactant free cellulose acetate membranes, Nalgene®)]. The culture was incubated ovemight at 37°C in a shaking incubator (225-300rpm). The following day, several 25ml cultures were set up using 500|xl o f ovemight starter culture, 0.2% maltose

Methods and Materials (w/v) and lOmM MgSO^ (sterilised by autoclaving). The cells were grown for 2-3 hours until an OD value between 1.0-2.0 at 600nm was obtained. At this OD there will be approximately 7.5 x 10^ cells/ml. The culture was cooled on ice and then centrifiiged at SOOOrpm for 10 minutes at 4°C (Heraeus Sepatech Megafuge l.OR). The supernatant was discarded and the pellet resuspended in 8ml o f ice cold lOmM M gS0 4 and mixed thoroughly. The resuspended cells were stored at 4°C and were used within a day or two o f preparation.

2.11.4.5 Preparation of genomic phage library for hybridisation

Ip l o f the phage library (>1.4 x 10^^ pfu/pl) was diluted in 299)il o f SM buffer. The diluted library was split into 3 aliquots o f lOOpl and 2.5ml o f freshly prepared competent X Ll-Blue MRA (P2) cells (see section 2.11.4.4) were added to each lOOpl aliquot o f library. Each aliquot o f library was plated directly onto LB agar mega plates and replica membranes were made. The mouse phage library was screened using standard hybridisation procedures with Denhardt’s reagent (see section 2.7.2 and 2.7.3). The probe was prepared as for the cosmid library screening. Positive plaques were identified, isolated and subjected to secondary and tertiary screening as described below.

The positive plaques were identified by aligning the X-ray autoradiograph with the master plate using the orientation marks. The positive plaques were then cored out using a sterile 1ml pippette tip, with the end cut off. The plaques were then resuspended in 500pl o f SM buffer and 20pl o f chloroform was added. The chloroform helps keep the DNA intact and prevents the growth o f fungi. The suspension was incubated at 4°C for a minimum o f 2 hours to enable the phage particles to equilibriate. Subsequently, 5 p i o f the suspension was diluted further in 500pl o f SM buffer and 50pl, lOOpl and 300pl aliquot o f this dilution was added to 500pl o f freshly prepared competent XL 1-blue MRA (P2) cells and incubated for 10 minutes in a 37°C oven. Each aliquot was added to 10ml pre-warmed (45°C) top agar and spread on to 137mm LB agar plates. One plate with an even spread o f plaques was selected to represent each positive plaque identified. Replica membranes were made, hybridised and subjected to secondary screen as described above. If the spread o f plaques was too concentrated, the original phage suspension was diluted by a further 100 fold serial dilutions and the same range o f

Methods and Materials volumes was spread out or vice versa.. The same process was repeated for the tertiary screen, in this case the plaques were plated onto 90 mm LB agar plates. W hen necessary, a fourth screening was performed.