Biomedical Engineering Reference
In-Depth Information
8. Heat shock the cells in 42 °C water bath for 45 s and
immediately add 80
μ
L of LB medium. Shake the reaction for
1 h at 37 °C.
9. Spread 100
L of the transformation mix on LB agar plates
containing the proper antibiotics (including 15
μ
g/mL chlor-
amphenicol for Gateway cassette selection) and incubate over-
night at 37 °C.
10. Pick 6-8 separate Destination vector colonies into Falcon 2059
culture tubes containing 2 mL of SB medium with antibiotic
and grow overnight at 37 °C with 200 rpm shaking.
11. Spin 1 mL of the culture in a microcentrifuge to pellet the
cells, and isolate plasmid using the FastPlasmid kit, eluting the
DNA in 50
μ
L of elution buffer.
12. Verify the size of the plasmid using agarose gel electrophoresis.
Load 1
μ
L of the purifi ed Destination vector DNA on a 0.8 %
agarose gel, and compare sizes to the Supercoiled DNA ladder.
13. Additional confi rmation of the Destination vector colonies
should be carried out by restriction enzyme analysis, using an
enzyme that will produce diagnostic bands to provide informa-
tion on the orientation of the Gateway cassette (
see
Note 23
).
In addition, sequence confi rmation across the Gateway cas-
sette boundaries is strongly suggested (
see
Note 24
).
14. Glycerol stocks of the
E. coli
strains containing Destination
vector clones should be made by adding 100
μ
μ
L sterile fi ltered
60 % glycerol to 300
L of culture. After mixing and incubat-
ing at room temperature for 5 min, these stocks can be frozen
at −80 °C and used to start new cultures if more Destination
vector is required in the future.
μ
4
Notes
1. It is a common problem for new Gateway cloners to confuse
the reading frames and directions of the “reverse”
att
sites
needed to make
attR
containing Entry clones. We strongly
recommend the use of in silico modeling of Gateway cloning
prior to ordering oligonucleotides. Several products can be
used for this purpose, including VectorNTI (Life Technologies,
Carlsbad, CA) and Clone Manager (SciEd Software, Cary,
NC). Many other programs can also be tricked into carrying
out Gateway reactions by pretending that the Gateway
att
sites
are restriction sites. In any case, it is always best to validate the
reactions in the computer before spending time and money on
incorrect sequences.
2. The addition of 3 % DMSO to the PCR can help to reduce the
effects of GC-rich primers or template DNA. Often it is not
