Analysis Of Genomic Lambda Libraries

The X ZAP II insertional mouse library filters were initially screened with lacZ, but this produced substantial background signal, so it was decided to use the p-actin probe which produced much cleaner results.

The X EMBL 3 Tg library filters were initially screened with the isolated Bank fragments, Tg710 and Tg300. They were subsequently screened with the P-actin probe and then the lacZ probe.

The X EMBL 3 wt library filters were screened with the isolated flank fragments, Tg710 and Tg300, only.

2.7.1 Plaque Hybridisation

Library filters were hybridised in plastic boxes using approximately 20ml of hybridisation buffer per library filter. For a library screening of 10 filters, 200ml of hybridisation buffer was required and consisted of:

60ml 20X SSC

10ml lOOX Denhardts

5ml 20% SDS

125ml Deionised water

This had salmon sperm DNA (ssDNA) added to a final concentration of O.lmg/ml; the ssDNA was boiled for 5 mins before being added to the pre-warmed

prehybridisation buffer (at 65°C). The library filters were added to this buffer, one- by-one, and incubated at 65°C with shaking for at least 1 hr. The probe that was to be used to screen the library was labelled (2.5.3), the final probe concentration being ~ 2ng/ml. The library filters were removed from the prehybridisation solution, briefly, and the labelled probe was added and mixed well. The filters were then returned to the “ hot” hybridisation buffer, one-by-one, and incubated at 65°C overnight with shaking. The filters were washed at a stringency of 0. IX SSC; 0.1% SDS at 65°C (2.5.5) and exposed to X-ray film at -70°C for 3-4 days. The “ hot” hybridisation buffer was saved at RT for subsequent screening hybridisations. 2.7.2 Screening Of The Libraries

The autoradiographs from the (A) and (B) filters were overlaid, with the orientation marks matching, and duplicate positive signals were noted. The filters were aligned over the original agar plates and the orientation marks lined up. Positives were cut out from the plates using a cut-off blue tip and the “ plug” was put into 1ml of SM buffer containing 5|il chloroform. These were allowed to elute at RT for 4 hrs and then stored at 4°C. The primary plug stocks were titred and the phage replated. A 1 in 1000 dilution was made of each primary plug stock (Iql stock in lOOOp.1 SM buffer) and 10, 2.5 and Ifil volumes were plated using lOOql plating cells (2.6.5). The plates were incubated overnight at 37°C. The appropriate dilution plate was chosen on the basis that it had a good number of plaques (-1000) which were not overlapping too much; the chosen plates were cooled at 4°C. Filter lifts were taken, with a duplicate, but this time small circular (90mm diameter) Hybond N-i- filters were used and they were marked with 4 orientation marks (2.6.7). The secondary filters were prehybridised in 100ml hybridisation buffer for at least 1 hr and then transferred to the pre-warmed “ hot” hybridisation buffer that was used in the primary screening. They were incubated overnight at 65°C with shaking. The

secondary filters were washed and exposed to X-ray film at -70°C overnight (2.7.1). Duplicate filters were aligned with the agar plates and a smaller plug was taken, corresponding to a duplicate positive signal. Plugs were eluted into 1ml SM buffer with 5|il chloroform at RT for 4 hrs. Using this secondary plug stock, a 1 in 1000 dilution was made and lp.1 plated out using lOOp.1 plating cells. The tertiary plates were incubated overnight at 37°C and then placed at 4°C to cool. Filter lifts were

taken from these plates, in duplicate, and hybridised with the ‘ ‘hot’ ’ hybridisation buffer. The tertiary filters were washed at a stringency of O.IX SSC; 0.1% SDS at 65°C and exposed to X-ray film at -70°C for ~ 5 hrs. Positive signals on the

autoradiographs of the tertiary filters now corresponded to single, isolated plaques. These plaques were picked into 1ml SM buffer with 5|ll chloroform and stored at 4°C to allow the phage to elute. The tertiary plug stock was used to prepare plate lysates which were either used as high titre phage stock or for lambda DNA isolation.

I. The X ZAP II Library

The X ZAP n library was screened with p-actin and produced 14 positives in the primary screening. The positives were reduced to 8 in the secondary screen, which were all isolated as single plaques. High titre phage stocks were made and DNA subsequently isolated.

II The X EMBL 3 Libraries

The wild-type (wt) X EMBL 3 library was screened with the isolated tlank

sequences, Tg710 and Tg300, and produced 2 positives in the primary screen that both came through to tertiary screening and subsequent DNA isolation.

The insertional mutant (Tg) X EMBL 3 library was screened initially with the flank sequences, Tg710 and Tg300, producing 12 positives in the primary screen that all came through to tertiary screening and subsequent DNA isolation. This library was also screened with P-actin and lacZ probes producing 86 and 84 positives,

respectively, in the primary screenings. No further work was done using these primary plug stocks.

2.7.3 Plate Lysate Preparation

The production of plate lysates is dependent upon producing plates that have

confluent plaque lysis. For each 9mm agar plate, lOOql tertiary plug stock and lOOql plating cells were combined and plated out using top agarose (2.6.5). Top agarose was scraped off confluent plates into a 13ml tube containing 3ml SM buffer. This was incubated at 37°C for 30 mins with shaking, centrifuged at 3000 rpm for 10 mins and the supernatant removed into a clean tube. A further 2m 1 of SM buffer was added to the top agarose, mixed and centrifuged at 3000 rpm for 10 mins. This supernatant was combined with the previously collected supernatant and centrifuged

at 3000 rpm for 10 mins. The resultant supernatant, the plate lysate, had chloroform added to 0.3% and was stored at 4°C.

2.7.4 In Vivo Excision Of pBIuescript Plasmid From Lambda ZAP II Vector Lambda ZAP II was designed to allow in vivo excision and recircularisation of any cloned insert contained within the lambda vector to form a phagemid containing the cloned insert. Plate lysate stocks from the tertiary screening of the library were used to generate phagemids. In a 50ml tube the following were combined:

200|ilX Ll-Blue cells (ODeoo = 1.5)

200|il Plate lysate stock (negative control had no phage added) l|il R408 helper phage

These were incubated at 37°C for 15 mins and 3ml 2X YT media was added and incubated at 37°C for 2.5 hrs with shaking. The tubes were then heated at 70°C for 20 mins and centrifuged at 3000 rpm for 5 mins. The supernatant was poured off, carefully, into a sterile tube. This stock contained the pBIuescript phagemid as filamentous phage particles and was stored at 4°C. Each rescued phagemid was used to set up the following:

Tube (1) 50|il of the phagemid stock and 200pl of XL 1-Blue cells

Tube (2) 10|il of a 1/100 dilution of the phagemid stock and 200pl of X L l- Blue cells

These were incubated at 37°C for 15 mins, plated out onto LB agar plates containing ampicillin at a final concentration of 50|ig/ml, inverted and incubated at 37°C

overnight. Plates with undiluted phagemid stock produced many colonies whilst the 1 in 100 dilution produced fewer, so it was possible to pick single colonies from the latter plates. The bacteria infected with helper phage alone did not grow because they did not contain ampicilhn resistance. A single colony was used to inoculate LB broth (plus ampicillin) and treated as a standard plasmid mini-prep (2.2.3-I). For long term storage, glycerol stocks were made and stored at -70°C (2.1.3).