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Contents 1 (Multi) Site Directed Mutagenesis 1.1 Oligonucleotide design 1.2 Mutagenesis Extension-Ligation Reaction 1.3 DpnI Digestion

(Multi) Site Directed Mutagenesis

Oligonucleotide design

The primers should be designed so that the desired mutation occurs at the exact center of the primer with ~15 bp of matching sequence on each side. For multisite mutations, do not design oligonucleotides that overlap each other; if you want to make multiple mutations within ~30 bp of each other, consider incorporating them in the same mutagenic primer or perform multiple rounds. Only forward primers are required, and all primers must be the same direction. All mutagenic primers should be 5’ phosphorylated (either during synthesis or enzymatically).

Mutagenesis Extension-Ligation Reaction

Assemble the following reaction: 1 x Phusion HF Buffer, 0.1 % Tween-20, 0.2 mM each dNTP, 3% DMSO, 0.5 mM NAD+, 2 ng/µL template, 350 nM of each mutagenic primer (for 1-3 primers, decrease to 175 nM for 4+ primers), 0.3 U/µL Ampligase, 0.02 U/µL Phusion Polymerase. Cycle the reaction at 98 °C for 30s, then 30 cycles of 98 °C for 15s, 55 °C for 60 s, 72 °C for 30s/kb, then hold at 72 °C for 10 minutes.

Example, for a 3-site 25 µL Reaction:

Reagent	Amount (µL)
5x Phusion HF Buffer	5
1 % Tween-20	2.5
Water	9.25
10 mM dNTP	0.5
DMSO	0.75
NAD+ (50 mM)	0.25
Primer 1 (6 µM)	1.5
Primer 2 (6 µM)	1.5
Primer 3 (6 µM)	1.5
Ampligase (5 U/µL)	1.5
Phuison (2 U/µL)	0.25
Template (50 ng total)	0.5
Total	25

DpnI Digestion

Add DpnI to 0.4 U/µL (0.5 µL for a 25 µL reaction), incubate for a minimum of 3 hours.

DpnI Digested product is ready for transformation. Transform 3 µL.