The cells were mixed by gentle flicking of the tubes. Each ligation reaction, 2C1L, was

added to a 1.5 mL microcentrifuge tube on ice, followed by 50 CIL of cells (100 CIL were

added to the TC). The tubes were mixed by gentle flicking and left on ice for 20 minutes.

The cells were heat-shocked by placing in a 420C water bath for 45-50 sec. The tubes

were then returned to ice for 2 minutes. S.O.C medium (Invitrogen Corp., Carslbad, CA),

950 C1L, was added to each tube (900 CIL was added to the TC). The tubes were then

incubated for 1.5 h at 370C with shaking (~150 rpm). The ampicillin/LB plates were

removed from 40C storage, 100 CIL of 100 mM isopropylthiogalactoside (IPTG, a P-

galactosidase inducer) and 20 CIL of 50 mg/mL 5-bromo-4-chloro-3 -indolyl-P-D-

galactoside (X-Gal, hydrolyzed by P-galactosidase to yield a blue product) were added,

and the mixture spread on each plate. The agar was allowed to absorb these compounds

for 30 min at 370C. Samples, 100 CIL, of each transformation culture were transferred to,

and streaked on, duplicate LB/ampicillin/IPTG/ X-Gal plates; for the TC, 20C1L of tube

culture was diluted with 180 CIL of S.O.C. medium, and 100 C1L of this dilution was

applied to the agar plates. The plates were incubated overnight (~16h) at 370C. Plates

were then stored at 40C for 30 minutes to facilitate color development. The white

colonies should contain plasmids with the insert, while the blue colonies do not contain

the insert since the protein-encoding sequence of the lac Z gene in the vector is not

interrupted by the insert and hence can lead to P-galactosidase synthesis and catalysis of

the X-Gal reaction.

 Colony PCR and culturing E.coli with insert of interest. Two white and one

blue colony from each plate were picked by a sterile wooden toothpick, which was

inserted in a PCR tube containing 5 CIL 10x PCR buffer, 5 CIL 10mM dNTP mix, 1 CIL