Quorum Sensing

electrophoresed at approximately 1 V/cm^2 , using TAE buffer,

until the dye front reach three-fourth of the agarose gel. The

DNA bands of interest are then excised from the gel using a

clean scalpel blade and cleaned up using a commercial kit

following manufacturer’s instructions.

3. The PCR products obtained in Subheading3.3,step 12, are
   cleaned up using a commercial PCR purification kit following
   the manufacture’s guidelines.


4. Both plasmid and PCR DNA concentration are determined by
   spectrophotometric analysis at A 260.


5. Cloning of antibodies heavy and light chains genes. The pri-
   mers OvJH(1-4)LINKFOR, OvVL(1-5)LINKBACK, and
   OvVK(1-2)LINKBACK, used for amplification of variable
   heavy (VH) and light chains (Vλor Vκ) from cDNA (seeSub-
   heading3.3,steps 9and 10 ), are designed to incorporate
   restriction sites AscI for heavy chain and MluI for light chain
   at the linker region. Equal quantities of purified VH PCR
   products are digested with 10 U of AscI perμg of DNA for
   3hat37C. Equal quantities of purified Vλor VκPCR
   products are digested with 10 U of MluI perμg of DNA for
   3 h at 37C.


6. After digestion, the DNA is purified by gel electrophoresis.


7. Equal quantities of purified VH DNA are independently
   ligated to Vλor to VкDNA by adding 150 U T4 ligase enzyme
   perμg of DNA and incubating the reaction mix at 37Cina
   water bath for 4 h. Into the reaction mix, 10 U each of AscI and
   MluI is added perμg of DNA to avoid the formation of VH-
   VH, Vλ-Vλ, and Vк–Vкproducts as successful ligation of VH-
   Vλand VH-Vкremoves AscI and MluI sites from the linker
   region.


8. After a 4 h ligation, 5μL of reaction product are run on a 1.5%
   (wt/vol) agarose gel and checked for successful ligation by a
   shift in band size from ~350 base pair to ~750 base pair.
   Successfully ligated DNA is purified by gel electrophoresis.


9. In order to incorporate cloning sites SfiI and NotI into VH-Vλ
   or VH-VкDNA fragments originated in thestep 8, set up PCR
   reactions as follows. Using 1 μL of purified VH-Vλ PCR
   product as template add 1μL (25 pmol) of primer mixture
   OvVH1BACK-Sfi, OvVH2BACK-Sfi, MuVH1BACK-Sfi,
   Hu4aBACK-Sfiand 1μL (25 pmol) of primer mixture OvJL1-
   FOR-Not, OvJL2FOR-Notfor VH-Vλtemplate. For VH-Vк
   template add 1μL (25 pmol) of primer mixture OvVH1BACK-
   Sfi, OvVH2BACK-Sfi, MuVH1BACK-Sfi, Hu4aBACK-Sfiand
   1 μL (25 pmol) of primer mixture OvJK1FOR-Not, OvJK2-
   FOR-Not, OvJK3FOR-Not. To each reaction mix add 25μL
   Phusion mix and 22μLofH 2 O. The reaction mix is heated up

340 Soumya Palliyil