Surface Modification of Cellulose Acetate with Cutinase and ...

Appendix I
Restriction enzyme(s) digestion of plasmid minipreps
(adapted from Sambrook et al., 1989)
Add 2 μl of appropriate restriction enzyme buffer (10x), 11.5 (or 11) μl of ultra pure
water, 0.5 μl restriction enzyme A (generally Roche 10 U/μl), (0.5 μl restriction enzyme
B (generally Roche 10 U/μl) for double digestions) and 6 μl of plasmid DNA miniprep
or midiprep. Incubate at appropriate temperature (37 ºC for most enzymes) for 3 h.
Analyse digestion fragments by gel electrophoresis.
Standard ligation of DNA fragments to plasmid vectores
(adapted from Sambrook et al., 1989)
Digest DNA fragment and plasmid vector with appropriate restriction enzyme(s). Purify
using QIAquick Gel Extraction Kit (QIAGEN).
To 50 ng of digested plasmid vector add digested DNA fragment enough to get a molar
vector:insert ratio of 1:5 (insert quantity (ng) = (insert size x 50 x 5)/ plasmid size). Add
1 μl ligase buffer (10x), 1 μl of ligase T4 1 U/ μl (Roche) and, if necessary, ultra pure
water to a final volume of 10 μl. Incubate overnight at 4 ºC.
Preparation and transformation of competent E. coli (XL1 Blue) SEM method
(adapted from )
Reagents
DMSO (Sigma)
IPTG (isopropyl-β-D-thiogalactopyranoside (Sigma)
40 mg/ml in sterilized destilled water
Luria-Bertani (LB)-Ampicillin agar
Tryptone 10 g/l
Yeast extract 5 g/l
NaCl 10 g/l
Agar 2%
Autoclave, cool to 50 ºC and add a stock solution of ampicillin 100
mg/ml to a final concentration of 75 mg/l, pour onto Petri plates.
LB medium (liquid)
Tryptone 10 g/l
Yeast extract 5 g/l
NaCl 10 g/l