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 reporter assays
The 3’UTR of mouse Cited2 (accession NM_010828.3, nucleotides 1051–1853) was amplified from mouse genomic DNA with Platinum Pfx DNA polymerase (Life Technology) and cloned into the pmirGLOW Dual Luciferase vector (Promega) downstream from the luciferase gene. The resulting constructs were sequenced to confirm their identity.

To modify the miR-145 target site in the Cited2-3’UTR, the plasmid was subjected to site directed mutagenesis using the Q5 Site directed mutagenesis kit (New England Biolabs) and the appropriate mutagenic primers (5’AATATGCTAACAGAGAAGATTAAACATGTGGGCCAAAC and 5’TGAAAACTTAAGTCTGTACTC). The miR-145 target site was changed from AACUGGAA to ACAGAGAA. The presence of the mutation was verified by sequencing.

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Manufacturer protocol

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

Download manufacturer protocol

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