2.1 Vector
Construction,
Site-Directed
Mutagenesis
1. Oligonucleotides.
2. DNeasy Plant Mini Kit.
3. pGEM-T easy vector system.
4. Restriction and modifying enzymes.
5. T4 DNA ligase.
6. 10Â T4 DNA ligase buffer: 400 mM Tris–HCl pH 7.8,
100 mM MgCl 2 , 100 mM DTT, 5 mM ATP.
7. Tango buffer: 33 mM Tris-acetate pH 7.9, 10 mM magnesium
acetate, 66 mM potassium acetate, 0.1 mg/mL BSA.
8. Agarose.
9. DNA molecular weight markers.
10. 6Â DNA-loading dye: 10 mM Tris–HCl pH 7.6, 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol,
60 mM EDTA.
11. TBE buffer: 89 mM Tris, 89 mM boric acid, 2 mM EDTA.
12. Luria-Bertani (LB) medium: 10 g/L Tryptone, 5 g/L yeast
extract, 10 g/L NaCl.
13. LB agar plates: LB supplemented with 1.5% [w/v] agar and
appropriate antibiotics (100 μg/mL ampicillin, 100 μg/mL
spectinomycin, or 50 μg/mL kanamycin).
14. Plasmid purification kit.
15. DNA fragment isolation kit.
16. Taq polymerase.
17. Phusion polymerase.
18. 100 mM dATP/dGTP/dCTP/dTTP: Prepare a 10 mM
dNTP mix from the 100 mM deoxynucleotide triphosphate
stocks.
19. LR Clonase II Plus enzyme.
20. Gateway entry vectors: pENTR11, pENTR2B, pENTR4.
21. Binary expression vectors: pBIN61, pK7WG2D, pK7WGF2
(see Note 1).
22. psiCHECK reporter plasmid.
23. E. coli strains TOP10 and DB3.1 (see Note 2).
24. Gel electrophoresis apparatus.
25. Power supply.
26. Heatable water bath.
27. Shaking incubator set to 37
C and 180–200 rpm.
28. Non-shaking incubator set to 37
C.
29. Microcentrifuge.
30. Thermocycler.
31. Nanodrop microvolume spectrophotometer.
434
Ka ´ roly Fa ´ tyol
Construction,
Site-Directed
Mutagenesis
1. Oligonucleotides.
2. DNeasy Plant Mini Kit.
3. pGEM-T easy vector system.
4. Restriction and modifying enzymes.
5. T4 DNA ligase.
6. 10Â T4 DNA ligase buffer: 400 mM Tris–HCl pH 7.8,
100 mM MgCl 2 , 100 mM DTT, 5 mM ATP.
7. Tango buffer: 33 mM Tris-acetate pH 7.9, 10 mM magnesium
acetate, 66 mM potassium acetate, 0.1 mg/mL BSA.
8. Agarose.
9. DNA molecular weight markers.
10. 6Â DNA-loading dye: 10 mM Tris–HCl pH 7.6, 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol,
60 mM EDTA.
11. TBE buffer: 89 mM Tris, 89 mM boric acid, 2 mM EDTA.
12. Luria-Bertani (LB) medium: 10 g/L Tryptone, 5 g/L yeast
extract, 10 g/L NaCl.
13. LB agar plates: LB supplemented with 1.5% [w/v] agar and
appropriate antibiotics (100 μg/mL ampicillin, 100 μg/mL
spectinomycin, or 50 μg/mL kanamycin).
14. Plasmid purification kit.
15. DNA fragment isolation kit.
16. Taq polymerase.
17. Phusion polymerase.
18. 100 mM dATP/dGTP/dCTP/dTTP: Prepare a 10 mM
dNTP mix from the 100 mM deoxynucleotide triphosphate
stocks.
19. LR Clonase II Plus enzyme.
20. Gateway entry vectors: pENTR11, pENTR2B, pENTR4.
21. Binary expression vectors: pBIN61, pK7WG2D, pK7WGF2
(see Note 1).
22. psiCHECK reporter plasmid.
23. E. coli strains TOP10 and DB3.1 (see Note 2).
24. Gel electrophoresis apparatus.
25. Power supply.
26. Heatable water bath.
27. Shaking incubator set to 37
C and 180–200 rpm.
28. Non-shaking incubator set to 37
C.
29. Microcentrifuge.
30. Thermocycler.
31. Nanodrop microvolume spectrophotometer.
434
Ka ´ roly Fa ´ tyol