Cells
An aliquot of the ligation batch (see section ligation) was added to 200
l competent cells, mixed and stored on ice for 30 minutes. Up to 40 ng of
DNA can be used for each transformation reaction. The mixture was transferred to
a water bath, preheated to 42psy176 C, for 2 minutes. After addition of 1 ml
LB medium, the cells were incubated at 37psy176 C for one hour without
shaking. This period allows the bacteria to recover and to begin to express
antibiotic resistance.
The medium was now spread onto selective media (see section amp), which
were treated with 20 l 100mM IPTG and 40 l Xgal before.
The cells were spread over the entire surface of the medium by moving a
sterile, bent glass rod back and forth gently over the agar surface. The glass
spreader was sterilized by dipping in 95% ethanol and then holding it into the
flame of a bunsen burner.
The plates were incubated overnight at 37psy176 C to allow plaque formation
and colour indicator reaction
. Plaques were picked within
the next 24 hours.