Quorum Sensing

(c) MuVH1BACK and mixture of OvJH1LINKFOR, OvJH2-

LINKFOR, OvJH3LINKFOR, OvJH4LINKFOR.

(d) Hu4aBACK and mixture of OvJH1LINKFOR, OvJH2-

LINKFOR, OvJH3LINKFOR, OvJH4LINKFOR.

10. To amplify ovine variable lambda and kappa chains, set up
    gradient PCR reactions using the following primer pairs:
    (a) OvVL1LINKBACK and mixture of OvJL1FOR and
    OvJL2FOR.
    (b) OvVL2LINKBACK and mixture of OvJL1FOR and
    OvJL2FOR.
    (c) OvVL3LINKBACK and mixture of OvJL1FOR and
    OvJL2FOR.
    (d) OvVL4LINKBACK and mixture of OvJL1FOR and
    OvJL2FOR.
    (e) OvVL5LINKBACK and mixture of OvJL1FOR and
    OvJL2FOR.
    (f) OvVK1LINKBACK and mixture of OvJK1FOR, OvJK2-
    FOR and OvJK3FOR.
    (g) OvVK2LINKBACK and mixture of OvJK1FOR, OvJK2-
    FOR and OvJK3FOR.


11. Each PCR reaction contains 1μL (25 pmoles) of each primer/
    primer mixture, 1μL cDNA fromstep 8,25μL2
    Phusion
    master mix, and 22μLofH 2 O. Heat up to 98C for 30 s and
    then start a 30 cycle temperature gradient program of denatur-
    ation at 98C for 10 s, annealing at gradient temperatures of
    52–72C for 30 s and polymerization at 72C for 30 s
    followed by a final extension of 72C for 10 min.


12. Check the PCR products by agarose gel electrophoresis. Mul-
    tiple reactions (about 12
    50 μL) should be performed for
    each primer pair using the optimum annealing temperature, in
    order to obtain a suitable quantity of DNA.

3.4 Construction of
Single Chain Variable
Fragment (scFv) Phage
Display Library

1. For small scale plasmid preparation, plasmid DNA is harvested
   from 3 mL of bacterial culture grown in Ampicillin
   (100μg/mL) containing medium using appropriate commer-
   cial kits following manufacturer’s instructions. For large scale
   plasmid preparation, DNA is extracted from 500 mL of bacte-
   rial culture grown under same conditions and using appropri-
   ate commercial kits following manufacturer’s instructions.


2. Gel extraction and purification of DNA. Plasmid DNA is elec-
   trophoresed on a 1% (wt/vol) agarose TAE gel containing
   ethidium bromide. A commercial DNA Molecular Weight
   Marker can be used for comparing the size of DNA bands. All
   samples are mixed (9:1 ratio) with 10
   DNA loading dye and

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