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7. QIAquick PCR Purification Kit (Qiagen).
8. Pfu DNA polymerase.
9. Acceptor plasmid DNA.
10. Antarctic phosphatase.
11. T4 DNA ligase.
12. DH5α competent E. coli cells (High Efficiency).
13. LB liquid and agar media containing appropriate antibiotic.
14. QIAprep Spin Miniprep Kit (Qiagen).
1. Acceptor plasmid DNA.
2. Donors’ DNA: either genomic DNA containing targeted
genes, or plasmids carrying the cloned targeted genes.
3. DNA primers for PCR reactions.
4. Pfu DNA polymerase.
5. QIAquick PCR Purification Kit (Qiagen).
6. AarI restriction enzyme.
7. 50× (0.025 mM) oligonucleotide containing AarI recognition
sequence.
8. AarI 10× reaction buffer.
9. Antarctic phosphatase.
10. 10× Antarctic phosphatase buffer.
11. T4 DNA ligase.
12. 10× T4 DNA ligase buffer.
13. DH5α competent E. coli cells (high efficiency).
1. Synthetic drop-out medium (liquid and solid).
2. Mutant plasmid library.
3. Round-bottom tubes with 35 μm cell strainer caps (BD).
4. 10 mM K. lactis α-factor pheromone.
5. 10 mg/mL cycloheximide.
6. Zymoprep™ Yeast Plasmid Miniprep II (Zymo Research).
3 Methods
1. Prepare a stock solution of Target DNA in water. The amount
of Target DNA depends on desired mutation frequency (see
Table 1). As an example we use the yeast G protein-coupled
receptor Ste2.
2. Prepare a 50 μL reaction as follows:
2.2 AarI Mediated
Domain Shuffling
2.3 Yeast-Based
Sorting of Rewired
Pathway Interactions
3.1 Error-Prone PCR
Raphaël B. Di Roberto et al.