Biomedical Engineering Reference
In-Depth Information
the one used for cloning of level −1 modules. Additionally, the
vector may contain a lacZ
fragment to allow blue-white selection
of the resulting clones. For MoClo vectors, two restriction sites
for a second type IIS enzyme (BsaI) are placed fl anking the two
fusion sites to allow the level 0 modules to be further subcloned
(for assembly of transcription units, for example). If the assembled
modules do not need to be subcloned, restriction sites for this
second enzyme may be omitted.
α
3.6 Golden
Gate Assembly
Once entry constructs and the recipient vector are made and
sequenced, assembling the fragments only requires pipetting all com-
ponents into a reaction mix and incubating the mix in a thermal cycler.
1. A restriction-ligation is set up by pipetting into a tube 40 fmol
(approximately 100 ng,
see
Note 7
) of each level −1 module
(or PCR fragment) and of the vector, 2
μ
L 10× ligation buffer,
10 U (1
μ
L) of BpiI, and either 3 U (1
μ
L) of ligase for assem-
bly of 2-4 modules or 20 U (1
L) HC ligase for assembly of
more than 4 modules (fi nal volume of 20
μ
L).
2. The restriction-ligation mix is incubated in a thermal cycler.
For assembly of 2-4 level −1 modules, incubation for
60-120 min at 37 °C is suffi cient. If more modules are ligated
together, the incubation time is increased to 6 h, or cycling is
used as following: 2 min at 37 °C followed by 3 min at 16 °C,
both repeated 30-50 times (
see
Note 8
).
3. Restriction-ligation is followed by a digestion step (5 min at
37 °C for BpiI or 50 °C if BsaI is used for cloning) and then by
heat inactivation for 5 min at 80 °C. The fi nal incubation step
at 80 °C is very important and is needed to inactivate the ligase
at the end of the restriction-ligation. Omitting this step would
lead to religation of some of the insert and plasmid backbone
fragments when the reaction vessel is taken out of the thermal
cycler and would lead to a higher proportion of colonies
containing incorrect constructs.
μ
The entire ligation is transformed into chemically competent
DH10B cells (
see
Note 9
).
1. Thaw frozen chemically competent cells (100
3.7 Transformation
of the Constructs into
Competent Cells
L per tube) on ice.
2. Add the entire ligation to the cells, and incubate on ice for
30 min.
3. Incubate 90 s at 42 °C in a water bath.
4. Let the cells recover on ice for 5 min.
5. Add 1 mL of LB medium to the cells, and incubate the tube at
37 °C in a shaker-incubator (150 rpm) for 45 min to 1 h.
6. After incubation, plate 25-100
μ
L of the transformation on
LB agar plates containing antibiotic and X-gal.
μ
