QuikChange Site-Directed Mutagenesis Kit, 10 Rxn

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- XL10-Gold cells are resistant to tetracycline and chloramphenicol. If the mutagenized plasmid contains only the tetR or camR resistance marker, an alternative strain of competent cells must be used.	- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

Upstream tips
- XL10-Gold cells are resistant to tetracycline and chloramphenicol. If the mutagenized plasmid contains only the tetR or camR resistance marker, an alternative strain of competent cells must be used.
Protocol tips
- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

Publication protocol

Plasmid Constructs. The β-arrestin-1 and β-arrestin-2 genes were searched for GREs and nGREs using Sequence Motif Search (www.genome.jp) and DNA Pattern Find (4). Fragments from the β-arrestin-1 promoter, β-arrestin-1 intron-1, β-arrestin-2 promoter, and β-arrestin-2 intron-11 were amplified from A549
cell genomic DNA using the FastStart High-Fidelity PCR System (Roche). The β-arrestin-1 promoter (∼1.7 kb) was cloned into pGL3-Basic (Promega) to generate βarr1-Pro using the following
primers: 5′-GTTCAGGCTAGCGTCCGCGACGGTCGCAGGGAGGTC-3′ and 5′-GTTCAGGGTACCGTGGAGAGTGGCAGGACCTGGCTTCACC-3′. The β-arrestin-2 promoter (∼1.0 kb) was cloned into pGL3-Basic to generate βarr2-Pro
using the following primers: 5′-GTTCACGGTACCTTCTGCGCATCCTTCAGAAAGACTGTCCTC-3′ and 5′-GTTCACAGATCTTCGGTTCGCGGCTCGCTCGCAGC-3′. The β-arrestin-1 intron-1 fragment (∼500 bp) was cloned into pGL4.23 (Promega) in the forward and reverse orientations to generate intron1F-GRE and intron1R-GRE, respectively, using the following primers: 5′-GTTCAGGGATCCTGCAGTCCCTCCTATGTGGACAGG-3′ and 5′-GTTCAGGGATCCGCTGTTGAACCACTGAATGG-3′. The intron-1 GRE sequence GGAACAnnnAGTTCT was mutated to GGAAGGnnnAAGTAT
using the QuikChange site-directed mutagenesis kit (Stratagene) to generate intron1F-mut and intron1R-mut. The β-arrestin-2 intron11 fragment (∼800 bp) was cloned into pGL4.23 in the forward and reverse orientations to generate intron11F-nGRE and intron11RnGRE, respectively, using the following primers: 5′-GTTCGTAGATCTCAGCAGGTTTAGTGACAAGTATGAAGGAG-3′ and
5′-GTTCGTAGATCTGAGAGGCATCGTGAAGAGGACGACGAC-3′. The β-arrestin-2 intron-11 fragment (∼800 bp) was
subcloned into βarr2-Pro in the forward and reverse orientations to generate Pro-intron11F-nGRE and Pro-intron11R-nGRE, respectively. The intron-11 IR1nGRE sequence TTCCnGGAGA was mutated to TGAAnAAAGA using site-directed mutagenesis to generate Pro-intron11F-mut1. The intron-11 IR1nGRE was mutated as above, and the IR0nGRE sequence ATCCGGAGA was mutated to AGAAAAAGA using site-directed mutagenesis to generate Pro-intron11F-mut2. The sequence of all constructs was confirmed by DNA sequencing.

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