...production, including codon optimization, deletion of the C-terminal 18-21 amino acids and a D614G change which...reporter vector expressing firefly luciferase with neomycin resistance. Other Resources Protocols Bloom lab...

...expression vector. All His-tags are cloned at the C-terminus of MDH and, if indicated on the plasmid map...Organism or species (e.g., human, watermelon, Streptomyces ) Subcellular compartment (chloroplastic, cytoplasmic...

...at 37°C for 12-18 hr in a shaking incubator. Note: Some plasmids or strains require growth at 30°C. If ...more slowly. Also, bacteria incubated at 30°C rather than 37°C often require longer incubation times. Double...Chloramphenicol 25 µg/mL Coumermycin 25 µg/mL Gentamycin 10 µg/mL Kanamycin 50 µg/mL Spectinomycin 50 µg/mL Tetracycline...

...gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish Xenopus CRISPR Resources ...and Port pAc-sgRNA-Cas9 dU6 BspQI Transfection Puromycin yes, cut Ji-Long Liu U6-BbsI-crRNA dU6 BbsI Transfection...

...doi.org/10.1371/journal.pone.0197185. Chamness, James C., et al. “An Extensible Vector Toolkit and Parts Library...Toolkit for Extensive and Flexible Engineering of Saccharomyces Cerevisiae.” ACS Synthetic Biology, vol. 12,...

.... Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, Rooney DL, Zhang M, Ihrig MM, ... 11586 pSico PGK puro Cre addition causes the puromycin gene to be recombined out of the construct, activating...12084 pSicoR PGK Puro Cre addition causes both puromycin and shRNA to be recombined out of the construct...Derivative of pMKO.1 puro with GFP instead of puromycin resistance gene William Hahn Additional Resources...

...heat-inactivated FBS and 5mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and FBS lots can promote...it can be inactivated in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health...dose of antibiotic, which may not be stable at 37 °C. To achieve a stable cell pool, the antibiotic selection...well plate and left undisturbed for 13 days. (a, b, c) Colonies formed by expansion of single cells for ...deliver mammalian antibiotic resistance (e.g., puromycin, blasticidin), which enables selection of a stable...preparation Appropriate antibiotic for selection (e.g. puromycin, blasticidin) Reagent Preparation DMEM Complete...obtaining accurate results. Check the cells for mycoplasma (Link opens in a new window) regularly Do not...

...involves co- expression of a Cas9 protein bearing a C terminus SV40 nuclear localization signal with one...plasmids is described in: Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Dicarlo...meningitidis Cas9 (NM) + tracrRNA expression, cloDF13/spectinomycin 48668 M-SPcas Mammalian S. pyogenes Cas9 expression...pyogenes Cas9 (SP) + tracrRNA expression, cloDF13/spectinomycin 48647 DS-ST1cas Bacterial S. thermophilus #1...#1 Cas9 (ST1) + tracrRNA expression, cloDF13/spectinomycin 48648 DS-TDcas Bacterial T. denticola Cas9 (... (TD) + tracrRNA expression, cloDF13/spectinomycin 48649 PM-SP!TA Bacterial SP crRNA expression: targets...

...Constitutive 2 Patrick 105922 pAAV-CBh-mKate2-IRES-MCS (C-gap-WT) CBh mKate2 Constitutive 2 Patrick 114469 pAAV-CaMKIIa-mCherry...dependent 8 Deisseroth 98927 pENN.AAV.CAG.Flex.GFPsm_myc.WPRE.SV40 CAG GFPsm_myc (does not fluoresce) Cre ...

...inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote...it can be inactivated in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health...obtaining accurate titers. Check the cells for mycoplasma (Link opens in a new window) regularly Do not... after 20–30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and... . The following day, cells were treated with puromycin. Colonies were allowed to grow in the presence...

...heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tips Different brands and lots of FBS can promote...it can be inactivated in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health...obtaining accurate titers. Check the cells for mycoplasma (Link opens in a new window) regularly Do not... after 20–30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and...

...Is Mediated by a Central DNA Flap. Zennou V, Petit C, Guetard D, Nerhbass U, Montagnier L, Charneau P. ... genomes have selectable markers, such as the puromycin resistance gene, conferring antibiotic resistance...