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7. Place reactions in thermocycler, using the program depicted in
Table 3:
8. Combine amplification PCR reactions and purify using a PCR
purification kit.
9. Digest >4 μg of purified amplification PCR product, and >4 μg
of desired acceptor plasmid with appropriate restriction
endonuclease(s). See Subheading 3.2 step 7 for recommended
AarI digestion protocol.
10. Dephosphorylate the ends of the linearized acceptor plasmid
by incubation with Antarctic phosphatase at 37 °C for 1 h to
reduce self-ligation background.
11. Purify digested amplification PCR product and acceptor plasmid with a PCR purification kit. Elute with no more than
30 μL water or provided elution buffer.
12. Ligate purified digested products using T4 DNA ligase. The
number of ligation reactions to perform depends on the desired
size of the library (see Table 4). Use 150 ng digested acceptor
plasmid and a 3 molar excess of digested amplification PCR
product per 20 μL reaction. Incubate at 16 °C overnight.
Table 3
PCR cycling protocol for amplifying error-prone PCRs with Pfu DNA
polymerase
Segment
Number of cycles
Temperature
Duration
1
1
95 °C
2 min
2
30
95 °C
30 s
Primer T m —5 °C
30 s
72 °C
2 min/kb
3
1
72 °C
10 min
Table 2
PCR cycling protocol for error-prone PCR with mutazyme
Segment Number of cycles Temperature
Duration
1
1
95 °C
2 min
2
20–30, see Table 1 95 °C
30 s
Primer T m —5 °C
30 s
72 °C
1 min (<1 kb
targets) or 1 min/
kb (>1 kb targets)
3
1
72 °C
10 min
Raphaël B. Di Roberto et al.