Transformation protocol

1. The supercompetent cells were thawed on ice. Epicurian coli XL-blue supercompetent cells (200236, Stratagene Cloning systems, California) with efficiency (cfu/pg of PUC 18 DNA) >1x10^ were used.

2. The cells were gently mixed by hand. About lOOjil of the supercompetent cells were aliquoted into each of two prechilled 15-ml Falcon 2059 polypropylene tubes. (One tube was for the experimental transformation and the other was for the control transformation.)

3. About l.Tpl of p-mercaptoethanol provided with the Stratagene 200236 Epicurian supercompetent cells kit or 1:10 dilution (from a 14.2 M stock solution) was added to each of the two aliquots of bacteria, giving a final concentration of 25 mM in each tube.

4. The contents of the tubes were swirled gently. The cells were incubated on ice for 10 minutes, swirling gently every 2 minutes.

5. One p,g of pComb3H % library DNA was added to one of the aliquots of cells and swirled gently. As a control, 1 pi of the pUClS control plasmid was added to the other aliquot of the cells and swirled gently.

6. The tubes were incubated on ice for 30 minutes.

7. The tubes were heat pulsed in a 42°C water bath for 45 seconds. 8. The tubes were incubated on ice for 2 minutes.

9. About 0.9 ml of preheated (42°C) SOC medium was added and the tubes were incubated at 37°C for 1 hour with shaking at 225-250 rpm.

10. Ten mis of prewarmed (37°C) superbroth(SB) (20pg/ml carbenicillin) and lOpg/ml tetracycline was added and immediately transformants were tittered by

plating lOOpl, lOpl and Ipl for test ligation and 100, 50, 10, 1, 0.1 ^il for library ligation on LB plates(100pg/ml carbenicillin (carb)).

11 The 10ml culture was incubated for 1 hr at 37°C on a shaker (300 rpm in all remaining steps). Following addition of carbenicillin to a final concentration of SO^ig/ml ,the culture was incubated for an additional hr at 37°C.

12. Helper phage VCSM13 (total of 10^^ pfu) usually ~ 1ml was added . The culture was transfered to a 100ml SB with 50pg/ml carb and lOpg/ml Tet and incubated on shaker for 2 hrs at 37°C.

13. Kanamycin was added to make a concentration of 70pg/ml and the culture was incubated on a shaker overnight at 30-37°C.

14. The cells were spun down at 4000 rpm (JAIO rotor), 4°C for 15 minutes. The supernatant was transferred to a clean bottle and 4% (w/v) PEG-8000 and 3% (w/v) NaCl were added. The supematent was placed on a shaker for 5 minutes to dissolve. The cell pellet was saved.

15. The phage was precipitated on ice for 30 minutes.

16. The precipitate was spun down (9,000rpm(JA 1 Orotor) for 20 minutes at 4°C) and the supernatant was discarded. The bottle was allowed to drain for 10 minutes on a paper towel to remove as much PEG as possible.

17. The pellet was resuspended in 2ml TBS/1 % BSA and centrifuged for 5 minutes at 14,000 rpm (Eppendorf microcentrifuge 5415C) and the supernatant was stored at 4°C (for long term storage 0.02% NaNg was added). Packaged phagemid preparations were used for panning only if they were freshly prepared, as proteases present in trace levels cleave Fabs from the

phage surface. Stored preps were reamplified prior to use in the panning protocol, ie step 9 of the panning protocol.

2.3.5 Panning

A panning experiment is made up of several rounds of recognition and replication. During each round, specific binding clones were selected and amplified. These clones predominated after three to five rounds.

1. High binding flat bottom 96 well ELISA plate (Costar, Coming, New York) wells were coated for 1 hour at 37°C with 25 pl/well of antigen (40pg/ml in O.IM NaHC0 3,pH 8.6) solution ( anti-idiotype 8.12 antibody) in coating

buffer in PBS, pH 7.5. Coating may be performed also at 4°C overnight. Generation of the 8.12 anti-idiotypic monoclonal antibody (mAB) has been published elsewhere (Livneh et al 1987b). A mouse was immunized with DNA/antiDNA antibody complexes that had been precipitated from the serum of one lupus patient and the anti-8.12 mAB was obtained by standard hybridoma methodology. The anti-8.12 mAB is purified from mouse ascites fluid by preparative IBP electrophoresis ( Davidson et al 1989, Paul at al 1991).

2. The fluid was shaken out of the wells and the plate was washed twice with deionized H2O.

3. The wells were blocked completely with 3% BSA TBS and the tray was sealed making it air tight or incubated in a humidified container. Incubation was done for 1 hour at 37°C, then the fluid was shaken out as above.

4. About 50 pi of phage suspension was added to each well ( total of about 10^^ pfu).

5. Incubation was undertaken for 2 hrs at 37°C after sealing the plate (or placing in a humidified container).

6. The phage was removed, the wells were filled with TBS/0.5% Tween 20 (TEST) and pipetted vigorously up and down. After 5 minutes , the TEST was removed. In the first round, washing was done once, in the second round the washing was done 5X times, and in the third and subsequent rounds lOx times.

7. The phage was eluted with 50 pil of elution buffer. (O.IM HCl) (adjusted with glycine to pH 2.2)/ESA Img/ml per well. Incubation was allowed for 10 minutes at room temperature, then pipetted up and down vigorously. The eluate was removed and neutralized with 3 nl of 2M Tris base per 50 ^il of elution buffer used.

8. About 2ml (per panning well) of fresh XL-1 blue (ODeoo ) grown with (lO^ig/ml) tetracycline were infected with the eluted phage and incubated at room temperature for 15 minutes. ( For reamplification of an existing library, 20^1 of PEG precipitated phage was added to 10ml of fresh XLs and the following steps were undertaken.)

9. About 10ml of 37°C prewarmed SE (20^ig/ml carb and 10 gg/ml tetracycline) was added and immediately the eluted phage was titered by plating 10, 1 and 0.1 \d on LE plates/carb. Incubation of the 12ml (or 20ml for reamplification) culture was performed for 1 hr at 37°C on a shaker.

10. To this culture the carbenicillin concentration was adjusted to 50gg/ml and incubated for 1 hour at 37°C on a shaker.

12. This was transferred to a 100ml SB culture with the same antibiotics and incubated on the shaker for 2 hrs at 37°C.

13. Kanamycin was added to make a concentration of 70^ig/ml and the culture was incubated on a shaker overnight at 30-37°C.

14. The cells were spun down at 4000 rpm (Sorvall ultracentrifuge), 4°C for 15 minutes. The supernatant was transferred to a clean bottle and 4% (w/v) PEG-8000 and 3% (w/v) NaCl was added and placed on a shaker for 5 minutes to dissolve.

15. The phage was precipitated on ice for 30 minutes.

16. The precipitate was spun down (9,000 rpm (Sorvall ultracentrifuge) for 20 minutes at 4°C) and the supematent was discarded. The bottle was allowed to drain for 10 minutes on a paper towel to remove as much of the PEG solution as possible.

17. The pellet was resuspended in 2 ml TBS/1 % BSA and, centrifuged for 5 minutes at 14,000 rpm (Eppendorf microcentrifuge ) and the supernatant was stored at 4°C (for long term storage , 0.02% NaN] was added). Packaged phagemid preparations were be used for panning only if they were freshly prepared, as proteases present in trace levels cleave Fabs from the phage surface. Stored phagemid preparations were reamplified prior to use in the panning protocol.

18. For subsequent rounds 50pl of the above packaged phagemid preparation was reapplied to antigen coated wells. The protocol was continued from step 5 as mentioned above.

19. The phage suspension was tittered by infecting 50pl volumes of XL-blue cells (ODôoo =0.5) with Igl of 10'^, 10'^ and 10'^ dilutions of phage suspension for 15 minutes at room temperature and plating on LB/carb plates (incubate overnight at 37°C).

2.3.6 Isolation of double stranded plasmid DNA from cell pellet of last