To sequence genes from the secondary screen, bacteria expressing clones from the ORFeome library were grown overnight in 2x TY media plus 100 μg/ml ampicillin; plasmids were extracted by using the PureYield Miniprep System (Promega, Wisconsin, USA). Purified plasmids were then quantified by using a NanoDrop 2000 spectrophotometer (Thermo-Fischer Scientific, Massachusetts, USA) and sent to Macrogen for sequencing (http://dna.macrogen.com/eng/ ).
Pureyield miniprep system
Manufactured by Promega
Sourced in United States
The PureYield Miniprep System is a laboratory equipment product designed for the isolation and purification of plasmid DNA from bacterial cultures. It provides a simple and efficient method for obtaining high-quality plasmid DNA samples, suitable for a variety of downstream applications.
Automatically generated - may contain errors
2 protocols using pureyield miniprep system
1
Plasmid Extraction and Sequencing
2
Recombinant CASR Mutant Expression
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Each CASR test variant (T699N, R701G, T808P) was introduced separately by site-directed mutagenesis (Stratagene, La Jolla, CA) into wild-type C-terminally FLAG-tagged human CASR cloned into the mammalian expression vector pcDNA3.1 (CaSR-FLAG/pcDNA3.1) as described previously [21 (link), 23 ]. Transfection-quality plasmid DNA was generated using the Pure Yield miniprep system (Promega Corp, Madison, WI) and quantitated using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA). HEK293 cells (RRID: CVCL_0045, https://scicrunch.org/resolver/RRID:CVCL_0045 ) were cultured as described previously [23 ] and 25 cm2 flasks of cells at approximately 60% confluency were transfected with 5 µg wild-type or mutant CaSR-FLAG/pcDNA3.1 (including a known inactivating control variant, L174R) or empty vector (pcDNA3.1) using Lipofectamine 2000 transfection reagent (Life Technologies, Invitrogen, Carlsbad, CA) prepared in antibiotic-free, OptiMEM medium (Invitrogen) as specified by the manufacturer. After approximately 6 hours incubation at 37 oC in 5% CO2, the transfection medium was replaced with Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and antibiotics and re-incubated until required for Western blotting, immunofluorescence, or IP-One assays.
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