Surface Modification of Cellulose Acetate with Cutinase and ...

Heat-shock the tubes in a thermoblock, at 42 ºC for 30 s with gentle agitation.
Incubate the tubes on ice for 10 min.
Add 800 μl of SOC medium and incubate the tubes for at 37 ºC for 1 hour with vigorous
shaking.
Spin cells down for a few seconds and discard about 950 μl of supernatant.
Ressuspend the pellet in the remaining 50 ul supernatant and plate on LB selective agar
plates. For blue-white colour screening add X-Gal 40 mg/ml to a final concentration of
40 μg/ml and IPTG 40 mg/ml to a final concentration of 40 μg/ml. Allow the plates to
dry before plating the transformation mixtures.
Incubate the plates at 37 ºC overnight.
Preparation of plamid DNA (Miniprep)
(adapted from Sambrook et al., 1989)
Reagents
Alkaline lysis solution I
0.2 M NaOH
1% (w/v) SDS
Alkaline lysis solution II
NaAc 3 M, pH 5.2
Ethanol 100% and 70% (v/v)
Plate 8 E coli colonies, per Petri plate, on LB selective agar. Incubate the plates
overnight at 37 ºC.
Resuspend ¾ of biomass, from each colony, in 200 μl of destilled water. Vortex for10
sec.
Add 200 μl of alkaline lysis solution I. Close the tubes tightly and mix the contents by
inverting the tubes rapidly for four times. Do not vortex.
Add 200 μl of alkaline lysis solution II. Close the tubes tightly and disperse alkaline
solution II through the viscous bacterial lysate by inverting the tubes rapidly for four
times.
Centrifuge the bacterial lysate at maximum speed for 5 min at 4 ºC. Transfer the
supernatant to a clean tube.
Precipitate nucleic acids from the supernatant by adding 500 μl of ethanol. Mix the
solution by inverting four times.
Collect the precipitated nucleic acids by centrifugation at maximum speed for 10 min at
4 ºC.
Remove the supernatant and add 500 μl of 70 % ethanol to the pellet. Centrifuge at
maximum speed for 5 min at 4 ºC.
Remove all of the supernatant and store the open tubes at room temperature until all the
ethanol has evaporated.