50 ng vector x a kb insert x 3 = ng insert required
 3.0kb vector 1
 where a is the approximate size of amplified insert

 Because of this limit in sample volume, the amount of insert actually used was less

than that recommended since the concentration of purified DNA was relatively low. The

ligation reactions were setup as follows (all volumes in CIL) in 0.5 mL tubes:

 Standard Positive Background
Component Reaction Control Control
2x Rapid Ligation Buffer, T4DNA ligase 5 5 5

pGEM T-Easy Vector (50 ng) 1 1 1

PCR-product (9 ng) 3----

Control insert DNA --- 2--

T4 DNA ligase (3 Weiss units/C1L) 1 1 1

DNase-free water 0 1 3

 The ligation buffer was mixed vigorously before use. The reactions were mixed by

pipetting and incubated for 1 hour at room temperature, followed by storing overnight at

40C.

 JM109 high-efficiency competent cells (>1x10s ofu/Clg DNA; Promega) were used

for transformation. The following procedure was performed using aseptic technique

(sterile tips and tubes, use of Bunsen flame to create upward convection in work area).

The tubes containing the ligation reactions were centrifuged for 1 minute at 10,000 rpm

and placed on ice. Another tube (transformation control, TC) was set up on ice; this

contained 0. 1 ng uncut plasmid (0. 1 CIL of 0. 1 mg/C1L solution used) in order to determine

the transformation efficiency of the competent cells. Tubes containing frozen aliquots of

JM109 cells were removed from -800C storage and placed on ice until thawed (~5 min).