Biomedical Engineering Reference
In-Depth Information
2.3 RF Cloning
Reaction and Analysis
1. Phusion high fi delity thermo-stable DNA polymerase, 5×
Phusion HF buffer, and dimethyl sulfoxide (DMSO) (New
England Biolabs, Ipswich, MA; Catalog # M0530S).
2. dNTPs set (Fermentas/Thermo Fisher Scientifi c, Glen-Burnie,
MD; Catalog # R0181). Prepare 10 mM dNTPs stock solu-
tion in ultrapure water.
3. Synthetic primers (forward and reverse) for RF cloning. Both
primers are diluted with TE buffer (10 mM Tris-HCl, 1 mM
EDTA, pH 8) or ultrapure water, to 25
2.3.1 RF Cloning
Reaction
μ
M stock solution (
see
Note 1
and Table
1
for primer design).
4. PCR product purifi cation kit (MEGAquick-spin Total, Intron
Biotechnology, Daejoen, South Korea; Catalog # 17286) or
equivalent.
5. Plasmid DNA purifi cation kit (QIAprep spin mini prep kit,
Qiagen, Hilden, Germany; Catalog # 27106) or equivalent.
6. Template DNA. If plasmid is used as a template dilute DNA
with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) or
ultrapure water, to 10 ng/
L stock solution.
7. Destination plasmid is diluted with TE buffer (10 mM Tris-
HCl, 1 mM EDTA, pH 8) or ultrapure water, to 10 ng/
μ
μ
L
stock solution.
8. Spectrophotometer (NanoDrop/Thermo Fisher Scientifi c,
Wilmington, DE; Catalog # ND-1000) or equivalent.
9. Thermocycler machine.
10. DpnI (New England Biolabs, Ipswich, MA; Catalog # R0176S)
or equivalent.
11. High effi ciency
E. coli
DH5
α
competent cells for heat shock
transformation (
see
Note 2
).
12. Sterile spreaders.
13. Ultrapure water purifi ed using Barnstead Nanopure ultrapure
water purifi cation system (Thermo Fisher Scientifi c Inc.,
Waltham, MA, USA) or equivalent.
14. Lysogeny Broth (LB) agar plates (10 g/L tryptone, 5 g/L
yeast extract, 5 g/L NaCl, and 12 g/L bacteriological agar)
supplemented with ampicillin (100
g/mL) and LB agar plates
supplemented with chloramphenicol (34
μ
μ
g/mL).
15. 37 °C incubator.
1. Synthetic primers for colony PCR and sequencing analyses
(
see
Table
1
).
2. Agarose gel electrophoresis apparatus.
3. Agarose.
2.3.2
Analysis
