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

Plasmid Construction
To generate different BiFC-fusion constructs of AR-FL, AR-V7, and ARv567es, we PCR amplified the AR-FL, AR-V7, and ARv567es cDNAs from their respective expression construct, and cloned the PCR amplicons separately into a TA-cloning vector (Promega). Fusion constructs of AR-FL, ARv567es, and AR-V7 with either VN or VC were generated by subcloning the cDNAs from the TA-plasmids into the SalI and XhoI sites of the pBiFC-VN155 and pBiFC-VC155 vectors. The mutant BiFC-AR-V and BiFC-AR-FL constructs with mutations at the FxxLF motif (F23,27A/L26A) and/or D-box (A596T/S597T) were generated by site-directed mutagenesis by using the Q5 site-Directed Mutagenesis Kit (New England BioLabs). BRET-fusion constructs of AR-FL, AR-V7, and ARv567es were generated by subcloning the AR-FL, AR-V7, and ARv567es cDNA from the respective TA-plasmids into the BamHI and XbaI sites of the pcDNA3.1-RLuc8.6 and TurboFP635 vectors (24). The doxycycline-inducible ARv567es lentiviral construct was generated by subcloning the ARv567es cDNA from its TA-plasmid first into the pDONR221 vector (Invitrogen) and subsequently into the doxycycline-inducible pHAGE-Ind-EF1a-DEST-GH lentiviral construct by using the Gateway Cloning System (Invitrogen). All plasmids were sequence verified.

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Papers

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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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Videos

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