Surface Modification of Cellulose Acetate with Cutinase and ...

SOB
TE buffer
Autoclave
Tryptone 2%
Yeast extract 0.5%
NaCl 10%
Autoclave, add sterilized MgCl2 to a final concentration of 20 mM.
PIPES 10 mM
CaCl2 5 mM
KCl 250 mM
Dissolve, adjust pH to 6.7 with KOH and add MnCl2 to a final
concentration of 55 mM, sterilize the solution by filtration and keep at
4 ºC.
X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)
40 mg/ml in dimethylformamide (DMF)
Preparation of competent E. coli (XL1 Blue)
Inoculate 250 ml SOB medium in a 2 L flask with 10 colonies;
Grow at 18 ºC with vigorous shaking (200-250 rpm) until an OD600 of 0.6.
Cool on ice for 10 min.
Spin cells down at 4ºC for 10 min at 2500 x g.
Resuspend cells in 80 ml of ice-cold TB buffer.
Cool on ice for 10 min.
Spin cells down at 4 ºC for 10 min at 2500 x g.
Gently resuspend pellet in 20 ml of ice-cold TB and add DMSO to a final
concxentration of 7 %.
Leave on ice for 10 min
Distribute into 200 ml aliquots (into sterile, ice-cold 1.5 ml eppendorf tubes) and freeze
in liquid nitrogen.
Store at -80 ºC.
Transformation of competent E. coli (XL1 Blue)
Thaw the competent E. coli cells on ice.
Add the experimental DNA (generally 1 μl of midi or mini and 10 μl of a ligation
reaction) to 200 μl of competent cells.
Mix gently and incubate on ice for 30 min.