Q5® Site-Directed Mutagenesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. - Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.

Protocol tips
- Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. - Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.

Publication protocol

To generate the deletion construct of Rictor, we separated the previously reported multiacetylation domain of Rictor into four parts (Δ1080–1089, Δ1089–1098, Δ1098–1110, and Δ1110–1128), according to predicted acetylated lysines within Rictor in silico (Fig. S2), and we performed the PCR-based construction of deletion. To create the 3KR and 3KQ mutants using the Myc-Rictor DNA plasmid, we replaced three predicted acetylation sites—K1116, K1119, and K1125—with R for the KR mutant and Q for the KQ mutant. We carried out site-directed mutagenesis using the Quick Change Kit (Stratagene) or Q5 Site-Directed Mutagenesis Kit (New England Biolabs).

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Manufacturer protocol

Download the product protocol from New England BioLabs for Q5® Site-Directed Mutagenesis Kit below.

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