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

DNA construction of WT and nonglycosylated SLC26 mutants.
Original cDNAs coding SLC26A1, A2, A3, A4, A5, A7, and A11 with COOH-terminal myc-DDK tag on plasmid pCMV6-Entry vector were purchased from Origene Technologies (Rockville, MD) and were used in these studies. The cDNAs encoding SLC26A2, A3, A6, and A9 in pGEMHE were obtained from Dr. M. F. Romero (Mayo), and the plasmids for SLC26A6 and A9 were used in these studies. The cDNA coding SLC26A8 in pcDNA3 (pRK5) vector was a gift from Dr. Gérard Gacon (Paris, France). These cDNAs encoding human SLC26 A1–11 WT, except A10, a pseudogene, were amplified by PCR using PfuUltra II Fusion High-Fidelity DNA polymerase (Aligent Technologies) and inserted into plasmid vector pcDNA3 (Invitrogen, Life Technologies) at NotI sites at both 5′- and 3′-ends. A new NotI site encoding three alanines was created just before the first methionine of SLC26A1, A2, A4, A5, A7, A8, and A11 to facilitate subcloning. A consecutive triple FLAG (DYKDDDDK) tag was created by PCR subcloning method at the NH2 terminus of all SLC26 family members. These clones did not include the COOH-terminal myc-DDK tag originally present in the Origene plasmids. Nonglycosylated mutants (N0) were created by site directed mutagenesis using Q5 site-directed mutagenesis kit (New England Biolaboratories) by mutating asparagine to aspartate (NxyzD, where xyz is the residue number mutated) in individual N-glycosylation acceptor sites (-N-X-S/T) and then in various combinations. All primers were synthesized by Integrated DNA Technologies. All DNA constructs were confirmed by DNA sequencing by ACGT (Toronto, Canada).

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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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