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

Luciferase Assay.
The luciferase coding sequence NANOG 3′-UTR reporter (cat. no. S807466) and luciferase coding sequence-only reporter (cat. no. S890005) were purchased from SwitchGear Genomics. Cells were seeded in a 96-well plate and transfected with luciferase-NANOG 3′-UTR reporter or luciferase-only reporter, and exposed to 20% or 1% O2 for 24 h. Luciferase activity was measured using a commercial kit (SwitchGear Genomics) and is presented as the activity ratio of luciferase-NANOG 3′-UTR to luciferase only. Adenine-to-thymine mutation of the luciferase-NANOG 3′-UTR reporter plasmid was performed by using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) according to the manufacturer’s instructions.

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Reviews

Discussion

Papers

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Manufacturer protocol

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

Download manufacturer protocol

Videos

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