46
linkers and linkers of specific sequences) can be appended to the 5′
end of the primers for the inverse PCR reactions. These linkers
between the two domains can be important for the switching property [5, 15, 16]; therefore, variable linkers and fixed composition
linkers are often tested.
2 Materials
1. QIAPrep Spin Miniprep Kit (Qiagen).
2. Invitrogen PureLink Gel Extraction Kit (Invitrogen).
3. DNA Clean and Concentrator Kit (Zymo Research Corp.,
Irvine, CA).
4. DNase I (Promega).
5. 0.5 M Tris–HCl pH 7.5.
6. 50% glycerol.
7. 10 mM MnCl 2.
8. 20 mg/mL BSA solution (100× stock, e.g., New England
Biolabs).
9. 1 M EDTA.
10. T4 DNA Polymerase (e.g., New England Biolabs, Ipswitch,
MA).
11. T4 DNA Ligase (2,000,000 U/mL) (e.g., New England
Biolabs, Ipswitch, MA).
12. 10 mM dNTPs each.
13. Antarctic Phosphatase (e.g., New England Biolabs,
Ipswitch, MA).
14. S1 Nuclease (e.g., Promega, Madison, WI).
15. 10× S1 Nuclease buffer (500 mM sodium acetate, 2800 mM
NaCl, 45 mM ZnSO 4 , pH 4.5).
16. Phusion High-Fidelity PCR Master Mix.
17. 1× TAE buffer: 40 mM Tris-base, 20 mM Acetic Acid, 1 mM
EDTA, pH 7.6.
18. Nanodrop.
19. Gene Pulser Electroporation Cuvettes (Biorad, Hercules,
California).
20. SOC medium: 2% Tryptone, 0.5% Yeast Extract, 10 mM NaCl,
2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 , 20 mM Glucose.
21. 245 × 245 mm Square Bioassay Dish (Corning, New York,
NY).
22. LB Agar:10% Tryptone, 5% Yeast Extract, 10% NaCl, and 15 g
Bacto Agar per 1 L of medium.
Lucas F. Ribeiro et al.