Biomedical Engineering Reference
In-Depth Information
Determine the DNA concentration spectrophotometrically.
Store the material at −20 °C until use.
4. Prepare 5 mL LB supplemented with the appropriate antibiot-
ics and cultivate
E. coli
DB3.1 cells containing the desired FX
cloning vectors overnight at 37 °C (
see
Note 12
). Isolate the
plasmids using a miniprep kit and determine the DNA concen-
tration. Store the material at −20 °C until use.
5. Mix 50 ng of an FX cloning vector with suffi cient PCR prod-
uct to have a vector:insert molar ratio of approximately 1:5 (
see
Note 13
). Add 1
μ
L 10× SapI-buffer and adjust the volume to
9
μ
L with UHP water. Subsequently add 1
μ
L SapI (2 U) and
incubate 1 h at 37 °C (
see
Note 14
).
6. Heat inactivate SapI for 20 min at 65 °C and allow the sample
to cool to room temperature. Add 1.25
μ
L 10 mM ATP and
L T4 DNA ligase (1.25 U) and incubate 1 h at room
temperature.
7. Heat inactivate the T4 DNA ligase for 20 min at 65 °C.
Transform 5
1.25
μ
L chemically com-
petent cells of an
E. coli
strain that is CcdB-sensitive (virtually
all
E. coli
strains lacking the F plasmid) and use 400
μ
L of the ligation mix to 100
μ
L medium
during the recovery phase. Plate 1 and 10 % aliquots on LB
agar supplemented with the appropriate antibiotic (
see
Note
15
). Incubate the plate overnight at 37 °C.
8. Pick a few single colonies from the plate to inoculate 5 mL LB
supplemented with the appropriate antibiotics (
see
Note 16
).
Incubate overnight at 37 °C. Isolate the plasmids with a mini-
prep kit and determine the DNA concentration. Verify the
insert by DNA sequencing. For inserts cloned into a pINITIAL-
derivative, primers INITforSQ and INITrevSQ can be used
(Table
1
). Store the vector at −20 °C until use. For inserts
cloned into an expression vector one can proceed as required
for the desired expression system.
μ
3.2 Subcloning of
Sequence- Verifi ed
ORFs from pINITIAL
to Expression Vectors
1. Prepare 5 mL LB supplemented with the appropriate antibiot-
ics and cultivate
E. coli
DB3.1 cells containing the desired FX
cloning expression vectors overnight at 37 °C. Isolate the plas-
mids using a miniprep kit and determine the DNA concentra-
tion. Store the material at −20 °C until use.
2. Mix 50 ng of an FX cloning expression vector with a sequenced
derivative of pINITIAL holding the insert of interest to have
an expression vector: sequencing vector molar ratio between
1:3 and 1:5 (
see
Note 12
). Add 1
μ
L 10× SapI-buffer and
adjust the volume to 9
μ
L with UHP water. Subsequently add
L SapI (2 U) and incubate 1 h at 37 °C.
3. Heat inactivate SapI for 20 min at 65 °C and allow the sample
to cool to room temperature. Add 1.25
1
μ
μ
L 10 mM ATP and
