...backbone contains a kanamycin resistance cassette, you would plate the cells on kanamycin-containing agar..... Only cells carrying plasmids that contain a kanamycin resistance cassette will grow. However, often ...successful uptake of your DNA of interest by taking advantage of bacterial characteristics such as prototrophy...strategy uses the toxin CcdB. Gateway cloning takes advantage of this property by including ccdB on its destination...

...different selection markers (blasticidin, neomycin, puromycin, hygromycin) and fluorescent protein reporters ... both of these cases, transposons combine the advantages of viruses and naked DNA while eliminating some...

...L-alanyl-L-glutamine (or alternative stable glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene...L-alanyl-L-glutamine (or stable alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add ...55 mL of heat inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots...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...

...deliver mammalian antibiotic resistance (e.g., puromycin, blasticidin), which enables selection of a stable...L-alanyl-L-glutamine (or alternative stable glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene...preparation Appropriate antibiotic for selection (e.g. puromycin, blasticidin) Reagent Preparation DMEM Complete...L-alanyl-L-glutamine (or stable alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add ... 55 mL of heat-inactivated FBS and 5mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and FBS...obtaining accurate results. 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...

... Targeting RNA Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs...1 none As Cpf1 Qi pKEE401 yes, cut S. pyogenes Neomycin Chen Do you have suggestions for other plasmids...

...yet they still share many of the same technical advantages that come with working with prokaryotes. This...primarily pertains to ORIs in budding yeast, Saccharomyces cerevisiae; however, we’ve also noted some features...required for the replication of fission yeast, Schizosaccharomyces pombe, vectors at the end.) In a previous...

...this protocol uses microscopy to determine the percentage of fluorescent cells, these cells can still be...L-alanyl-L-glutamine (or alternative stable glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene...L-alanyl-L-glutamine (or stable alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add ...55 mL of heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tips Different brands and ...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...assume one integration event per cell. When the percentage exceeds 40% you risk counting cells with multiple...

...contains 24 copies of SunTag, a synthetic scaffold that can recruit GFP fused to the SunTag-specific antibody...Imaging by Coat protein Knock-off) instead takes advantage of the ribosome’s movement. When the ribosome ...Using translation inhibitors cycloheximide and puromycin, Halstead et al. verified that translation is ...indicating translational repression. During later stages, single RFP-labeled osk and Oskar protein were ...scFv. Each time the reporter is translated, the SunTag sites on the nascent polypeptide recruit scFV-GFP..., as the authors of these papers did. Since the SunTag-based system tethers mRNAs to the plasma membrane...Additional Resources the Addgene Blog Learn More about SunTag Catch up on Your GFP Background Choose the Brightest...

...Voltron2 voltage sensors By Brian O’Neill New viral vector preps expressing the Voltron2 voltage indicator...-2 research New viral vector preps of Voltron2 voltage sensors AAV vectors for targeting microglia The... vectors for CROPseq-multi using selection by Puromycin, Zeocin®, or nuclear expression of BFP2.   ...earlier Voltron indicators (a fusion of Ace2N and HaloTag, Figure 4), they determined that an A122D mutation...ratio and better ability to record subthreshold voltage fluctuations, while having high fidelity during...neurons. The same A-to-D mutation in the related voltage indicator Ace2N-mNeon also led to similar improvements...negative-going sensors (with fluorescent signal at baseline voltage), the investigators found that administering these...

...NGAG and NGCG and SaCas9 with an NNGRRN PAM by mutagenizing the PAM-interacting domain. However, these alternative...mutants with PAM flexibility. In this system, the mutagenesis plasmid (MP6) permits quick, large-scale mutation...recognizing multiple PAMs should have a fitness advantage over proteins with single PAM recognition. After...bacteria where cleavage leads to the loss of spectinomycin resistance. xCas9 3.7 showed the broadest PAM...cell population measured with NGS) are shown as percentages, with fold changes compared to SpCas9 in parentheses...

...Wyatt and Dale Ramsden, UNC at Chapel Hill. One advantage to using the CRISPR/Cas system for genome engineering...Persistence of a Chromosome Break Diminishes Its Mutagenic Potential. PLoS Genet 5:e1000683 . https://doi.org...End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis. mBio 11: . https://doi.org/10.1128...

...that controls repression of gene expression in Saccharomyces cerevisiae. The system consists of dCas9, the... 2: The components of Casilio-ME1. Image from Taghbalout et al., 2019. Fusing epigenetic modifiers...

...HEKs. These systems built around HEKs offer an advantage to use the cell line and we will highlight some...cell lines are susceptible to contamination by mycoplasma and bacteria, but HEKs are also particularly ...

...the human gut bacteria B. thetaiotaomicron, Mycobacterium, Pseudomonas, and E. coli. However, many non-model...for CRISPRi transfer Mobile-CRISPRi offers two advantages over existing bacterial CRISPRi methods. First...

... Targeting RNA Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs...pyogenes URA3 Wyrick pCRISPomyces-2 61737 Bacteria BbsI yes, cut S. pyogenes Apramycin Zhao pRB1017 59936...none S. pyogenes Zon pCRISPomyces-1 61736 Bacteria BbsI yes, cut S. pyogenes Apramycin Zhao pMB70 47943 Worm...pyogenes tagRFP Ebert pL-CRISPR.SFFV.tRFP 57826 Mammalian/Lentiviral BsmBI yes, cut S. pyogenes tagRFP Ebert...activate, interfere, or nick. Selection , such as Puromycin or EGFP Cloning enzyme used for insertion of your...Mammalian/Lentiviral BbsI none S. pyogenes Puro, TagBFP Yusa lentiGuide-Puro 52963 Mammalian/Lentiviral...Mammalian/Lentiviral BsmBI yes, cut S. pyogenes tagRFP Ebert pFGC-pcoCas9 52256 Plant AscI, PacI, SbfI...

...majority of plasmids contain either an ampicillin or kanamycin resistance gene. Every step along the way involves...and prepare fresh media with antibiotics at each stage. The barcodes and LIMS help us track each sample...

...Properties of FLP Recombinase Evolved by Cycling Mutagenesis.” Nature biotechnology 16(7): 657–62. PubMed ...Sadowski. 1985. “The FLP Recombinase of the Saccharomyces Cerevisiae 2 Micron Plasmid Attaches Covalently...

... chromosomal aberrations. Pfeiffer P, et al. Mutagenesis (2000). PubMed. Multiple Pathways of Recombination...Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae. Paques F and Haber JE. Microbiology...

...GAL4 gene downstream of a tissue- or developmental stage-specific promoter and design a UAS reporter construct...genes for metabolic engineering applications in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197...

...carrying version 1 of the CellTag barcoding library. Version 1 of the Celltag library contains 19973 barcodes...carrying version 2 of the CellTag barcoding library. Version 2 of the Celltag library contains 4934 barcodes...carrying version 3 of the CellTag barcoding library. Version 3 of the Celltag library contains 5737 barcodes... genes along with 1000 non-targeting controls Puromycin Doench , Root 73179 Human gRNA pooled library ... genes along with 1000 non-targeting controls Puromycin Doench , Root Human activation pooled libraries...56,762 unique sgRNAs targeting 18,885 human genes Puromycin Doench , Root Human inactivation pooled libraries...human genes along with 500 non-targeting controls Puromycin Doench , Root 92386 Human CRISPR inhibition library...