Quick change

Manufactured by Promega

The Quick Change is a laboratory tool designed for rapid and precise site-directed mutagenesis. It allows users to introduce specific modifications into DNA sequences quickly and efficiently without the need for complex cloning procedures.

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In fusion cloning, by Takara Bio (1 mentions)

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Quick Change site-directed mutagenesis kit (4 citations) Quick Change mutagenesis kit (2 citations)

3 protocols using quick change

Cloning and Mutagenesis of Brr2p Domains

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Brr2p domains H2-Sec63-2 and Sec63-2 were polymerase chain reaction (PCR)-amplified and cloned into pBlueScript using SpeI/XhoI restriction sites. LexA-bait fusions of PRP2 and PRP16 were generated by InFusion cloning (Takara). The Gal4AD-prey fusion for H2-Sec63-2p was constructed in pACTII-stop using XmaI/BamHI sites. Mutations in PRP16 were created by site-directed mutagenesis (Quick Change, Promega).

Cordin O., Hahn D., Alexander R., Gautam A., Saveanu C., Barrass J.D, & Beggs J.D. (2014). Brr2p carboxy-terminal Sec63 domain modulates Prp16 splicing RNA helicase. Nucleic Acids Research, 42(22), 13897-13910.

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Constructing yeast strains for BRR2 and PRP16 studies

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A W303 brr2Δ strain was constructed by replacing the entire open reading frame (ORF) from one chromosomal copy of BRR2 with the kanMX6 cassette in W303 diploid cells (25 (link)), transformation with pRS316-BRR2 (URA3), sporulation and isolation of brr2Δ haploids carrying pRS316-BRR2. Mutant derivatives of pRS315-BRR2 were generated by site-directed mutagenesis (Quick Change, Promega) and their phenotypes analyzed after plasmid shuffle on 5-FOA to select for clones that had lost pRS316-BRR2 (26 (link)). 5-FOA-selected transformants were spotted on YPDA (yeast peptone, dextrose, adenine; complete, non-selective medium) agar and incubated for 2 days at 25°, 30°, 37°C or for 5 days at 18° or 15°C. W303 brr2Δ/isy1Δ was constructed by replacing the entire ISY1 ORF with natNT2 (27 (link)). W303 brr2Δ/prp16Δ was constructed by replacing one genomic copy of the entire PRP16 ORF with natNT2 in a diploid, heterozygous brr2Δ strain carrying pRS316-BRR2/PRP16, followed by sporulation and tetrad dissection. Mutant derivatives of a pRS314-PRP16 plasmid were constructed by site-directed mutagenesis. W303 brr2Δ/prp16Δ was co-transformed with pRS314-PRP16 and pRS315-BRR2 or mutant versions thereof for plasmid shuffle assays on 5-FOA to lose pRS316-BRR2/PRP16.

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Cloning and Mutagenesis of eIF3c Fragments

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Human eIF3c cDNA segments with defined ends were amplified by PCR using the oligos listed in Key Resource Table and cloned into pGBfusion1 (Reibarkh et al., 2008 (link)) after BamHI digestion to generate pGB-heIF3c plasmids (Key Resource Table). These plasmids were then used to produce in E. coli the heIF3c fragments employed in Fig. 1 and S1. The BN1 mutation (K380Q K383Q H386Q K389Q K391Q in Homo sapiens and R380Q K383Q H386Q K389Q K391Q in Drosophila melanogaster) was introduced by QuickChange™ (Promega) with oligos listed in Key Resource Table to the relevant h5MP1- or Dme5MP-expression plasmids (Key Resource Table). YEpL-GB-TIF5^B5-F and its BN1 mutant derivative were generated by subcloning into YEpL-TIF5-F (Key Resource Table) a 0.8-kb NdeI-SalI fragment encoding N-terminal GB1-fusion to Tif5p (aa. 201-405) WT or BN1 (Yamamoto et al., 2005 (link)), respectively, FLAG-tagged at its C-terminus. To produce the ACG firefly luciferase reporter plasmid p1785 (Key Resource Table), the complimentary oligos listed in Key Resource Table were annealed and inserted between the PstI and BamHI sites of pSV40 firefly Kozak ACG (p1525).

Singh C.R., Glineburg M.R., Moore C., Tani N., Jaiswal R., Zou Y., Aube E., Gillaspie S., Thornton M., Cecil A., Hilgers M., Takasu A., Asano I., Asano M., Escalante C.R., Nakamura A., Todd P.K, & Asano K. (2021). Human oncoprotein 5MP suppresses general and repeat-associated non-AUG translation via eIF3 by a common mechanism. Cell reports, 36(2), 109376.

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