to 98C for 30 s and subjected to two cycles of annealing and
polymerization using 65C as the annealing temperature for
VH-Vλand 64.4C as the annealing temperature for VH-Vк,a
further extension at 72C for 10 min follows. After 2 cycles of
initial amplification, the reaction mixes are subjected to a fur-
ther 25 cycles of PCR reactions with 72C as the annealing
temperature for both VH-Vλand VH-Vк. PCR products are
then purified using gel electrophoresis and extraction.
- For construction of the scFv phage display library, 10μg of the
phagemid vector pHEN 2a are digested with SfiI enzyme by
adding 20 U perμg of DNA and incubating at 50C for 4 h.
Afterwards, NotI enzyme is added to the reaction mix (20 U
perμg of DNA) along with a conversion buffer which increases
the NaCl concentration in the buffer to 50 mM and Tris–HCl
to 40 mM, which is optimum for the NotI reaction. The
reaction mix is incubated at 37C for further 4 h. The phage-
mid vector backbone is then purified by running the restriction
products on a 1% (wt/vol) agarose TAE gel, excising the DNA
band corresponding to ~3500 base pairs and extracting the
DNA by means of a commercial kit.
- The VH-Vλand VH-VкDNA with cloning sites incorporated
as described instep 9are digested with SfiI and NotI and
purified by gel electrophoresis. The VH-Vλand VH-Vкfrag-
ments (1μg of DNA each) are ligated into phagemid vector
backbone using T4 Ligase enzyme (400 U) with a vector:insert
ratio of 1:3. The ligation mixture is incubated at 16C for
16 h. Independent ligation reactions are set up for VH-Vλand
VH-Vкinserts. Ligated DNA is extracted with phenol/chlo-
roform, cleaned up using ethanol precipitation, and resus-
pended in 20μLofH 2 O.
- The ligated DNA fromstep 11is transformed by electropora-
tion intoE. coliTG1 cells by standard electroporation proce-
dures. Multiple electroporations are performed separately for
VH-Vλand VH-Vкligation reactions and collected into two
distinct pools (one for VH-Vλand one for VH-Vкligation
mixture) after recovery. 100μL of cells from each pool are
serially diluted tenfold in LB medium and plated onto TYE
plates containing 100 μg/mL Ampicillin and 1% (wt/vol)
glucose. The plates are incubated at 30C overnight and the
size of the phage display library is calculated from the titer
obtained from serial dilution plates. The rest of the cells are
plated onto large square TYE plates containing 100μg/mL
Ampicillin and 1% (wt/vol) glucose and incubated overnight at
30 C. After overnight incubation, the cells are scraped from
the bioassay dishes using 2TY medium and stored at 80 C
as 1 mL aliquots.
Monoclonal Antibodies Inactivating AHL Signal Molecules 341
