Biomedical Engineering Reference
In-Depth Information
10. Properly sized Entry clones should be sequence-verifi ed to
ensure that no oligonucleotide or PCR-generated errors have
been introduced (
see
Note 12
).
11. Glycerol stocks of the
E. coli
strains containing Entry clones
should be made by adding 100
μ
L sterile fi ltered 60 % glycerol
to 300
L of culture. After mixing and incubation at room
temperature for 5 min, these stocks can be frozen at −80 °C
and used to start new cultures if more Entry clone DNA is
required in the future.
μ
1. Add the following reagents to a microcentrifuge tube in the
order given (the total reaction volume should be 10
3.4 Multisite LR
Recombination
μ
L):
1-5
L H
2
O, each Entry clone DNA (50 ng,
see
Note 13
),
Destination vector DNA (100 ng,
see
Note 14
), and 2
μ
μ
L LR
Clonase II (
see
Note 15).
2. Incubate the reaction mixture overnight at 25 °C (
see
Note 16
).
3. Add 1
L of 2 mg/mL Proteinase K to inactivate the LR
Clonase II and incubate for 15 min at 37 °C (
see
Note 17
).
4. Add 1
μ
L of the LR II reaction to a microcentrifuge tube con-
taining 20
μ
L of chemically competent
E. coli
DH10B and
incubate on ice for 10-20 min (
see
Note 9
).
5. Heat shock the cells in a 42 °C water bath for 45 s and imme-
diately add 80
μ
μ
L of LB medium. Shake the reaction for 1 h at
37 °C.
6. Spread 100
L of the transformation mix onto an LB agar plate
containing the correct antibiotic (often ampicillin, but check the
Destination vector information) and incubate overnight at 37 °C.
7. Pick two separate colonies into Falcon 2059 culture tubes con-
taining 2 mL of SB medium containing the correct antibiotics
and grow overnight at 37 °C with 200 rpm shaking.
8. Spin 1 mL of the culture in a microcentrifuge tube to pellet the
cells, and isolate plasmid using the QIAprep Spin Miniprep
Kit, eluting the DNA in 50
μ
L of elution buffer (
see
Note 18
).
9. Verify the size of the plasmid using agarose gel electrophoresis.
Load 1
μ
L of the purifi ed Expression clone DNA on a 0.8 %
agarose gel, and compare sizes to the Supercoiled DNA ladder.
10. Additional confi rmation of the Expression clone should be car-
ried out by restriction enzyme analysis (
see
Note 19
). 1
μ
L of
Expression clone DNA can be digested using BsrGI restriction
endonuclease for 1 h at 37 °C.
11. Glycerol stocks of the
E. coli
strains containing Multisite clones
should be made by adding 100
μ
μ
L sterile fi ltered 60 % glycerol
to 300
L of culture. After mixing and incubating at room
temperature for 5 min, these stocks can be frozen at −80 °C
and used to start new cultures if more Expression clone DNA
is required in the future.
μ
