Una introducción a la historia material de CUBA

with a cDNA insert size range of 0.8 - 3.6 kb: Appendix 1) was used. Buffers, reagents and media are described in detail in Appendix 2.

2.5.1 Screening A P lacental cDNA Library

2.5.1.1 Storage and Preparation o f C om petent B acterial Cells for P hage Infection

E. coli (strain: Y1090) was stored freeze dried in vials and used to

prepare slopes for inoculation into TB medium (Appendix 2). 10 ml containing 100 pg/ml of ampicillin in sterile universals. The cultures were shaken overnight at 37°C.

2.5.1.2 P re p a ra tio n o f A gar P la te s U sed fo r S c ree nin g

Autoclaved agar (Appendix 2) was poured into sterile Petri dishes (Northumbria Biologicals) and allowed to set. On cooling the plates were inverted and allowed to dry before use. The poured plates were stored at room tem perature.

2.5.1.3 In fec tio n o f E .coli (strain : Y1090) W ith B a c te rio p h a g e (^ g tll) Two different Petri dish sizes (90 and 140 mm diameter) were used and their size determined the volume of infected bacterial culture and top agarose th at was used to accommodate phage plaques. A dilution of library in lambda phage diluent (Appendix 2) was used for infection th at would give an estim ated num ber of plaques per plate as illustrated in Table 2.4.

T able 2.4 The relationship of num ber of plaques to Petri dish diameter.

P e tri d ish d iam eter: 90 mm. 140 mm.

V olum e o f c u ltu re used: 200 pi 600 pi

V olum e o f to p ag aro se used: 3.3 mis. 9.9 mis.

E stim a te d n u m b e r o f p la q u e s1: 8,000 20,000

[Legend: u. The cDNA library was diluted to give the estimated number of pfu’s for the given size of Petri dish as described in section 2.5.1.]

The diluted library was incubated with bacterial culture in a w ater bath at 37°C for 20 minutes to allow infection to take place. Top agarose (Appendix 2) which had been equilibrated to 50°C was then added and mixed by gentle inversion. It was then swirled onto plates and allowed to set for 10 minutes.

2.5.1.4 T itra tio n o f T he L ib rary .

The library was diluted with lambda phage diluent in sterile capped polypropylene tubes to a 10*7 dilution. Then 10, 20 and 30 pi of the diluted

library was used to infect E. coli as described in Section 2.5.1.3. Inoculated

plates were inverted and incubated for 3 hours at 42°C, followed by 3 hours at 37°C. The num ber of plaques detected per plate is illustrated in Table 3.1 (Chapter 3).

2.5.1.5 The Ratio o f R ecom binant/Vector P hage in The Library.

A 10 pi aliquot of 10*6 dilution of library was added to the top agarose as described in section 2.5.1.3, except th at the top agarose also contained 9 mM iso-propyl-P-D-galactoside (IPTG) and 10 pM X-gal. The plates were incubated at 37°C overnight (Plate 3.2, Chapter 3).

2.5.1.6 Plaque Form ation.

Plates were prepared as in section 2.5.1.3. and incubated at 42°C for 3 hours, followed by 1 hour at 37°C. They were then overlaid with a notched/numbered 0.45 pm nitro-cellulose filter membrane th at had been impregnated (Appendix 2) with IPTG. The plates were further incubated for 3 hours at 42°C. The membranes were always handled wearing latex gloves and forceps were used to carefully remove the membrane overlay from the plate so as to avoid disturbing the top agarose lawn and lim it any tearing of the membrane. The membrane orientation was m arked on the plate and inverted plates were stored at 4°C.

2.5.1.7 B locking o f N itrocellulose O verlays and Im m unodetection.

Filters prepared as described in section 2.5.1.6 were placed back to back

in a plastic bag and 3 (9) mis of TBS-TB added for a 90 (140) mm filter. Air

bubbles were removed and the bag was sealed and left overnight at 4°C. The

filters were then washed three times for 10 m inutes each in TBS-T and

placed in a plastic bag with a 1:300 dilution of DAKO anti-PAPP-A in TBS-T for 3 hours at room tem perature. Following washing (as above) the filters were transferred into a plastic bag with a 1:100 dilution of biotinylated anti­

rabbit IgG (SIGMA Extra-3 kit, Appendix 1) in TBS-T for 1 hour at room

tem perature with rocking. They were then washed (as above) and sealed in

plastic bags with a 1:100 dilution of avidin-horseradish peroxidase conjugate

(SIGMA Extra-3 kit, Appendix 1) in TBS-T and incubated for 1 hour at room

tem perature with rocking. The filters were washed in TBS (3 times, 10

minutes each wash). Components (A and B, Appendix 2) of a chromogenic

substrate were prepared just before use and approximately 5 (15) ml were

applied to each 90 (140) mm filter. The colour was allowed to develop for

approximately 20 minutes. The filters were blotted dry and examined for coloured areas.

2.5.1.8 Iso la tio n o f P u ta tiv e R eco m b in an t P h ag e P laq u es.

Filters containing coloured areas (a positive immunoblot) were aligned with the Petri dish using the notch and filter outline on the dish as a guide. The agar/agarose containing the plaque th at corresponded to the coloured area was removed using a sterile Pasteur pipette tip. This agar plug was

placed into a sterile capped polypropylene tube and

vortexed with 50 pi of chloroform to lyse the bacterial cells. 1 ml of lambda phage diluent was then added and it was incubated for 1 hour at 37°C. The

lysate was diluted 103 fold and 50 pi of this was used to infect E. coli cells for

re-screening as described in section 2.5.1.3.

2.6 M aterials and M ethods Section for The Q uantitative