...DNA glycosylase inhibitor with a degradation tag (LVA tag) to achieve specific point mutations in E. coli... ideal number. First you generate four unique kanamycin-resistant plasmids, each containing a different...construct, you clone all but one of your gRNAs into spectinomycin-resistant plasmids pX330S-2 to pX330S-(last ...multiple promoter-gRNA cassettes, making them advantageous for small capacity vectors like AAV. In addition...monocot and dicot plants, with a choice of either hygromycin or Basta selection. Yang Lab Single Transcript.... Yin, Linlin, et al. “Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs...

...cells according to whether or not they have been tagged with a fluorescent protein or dye. It separates... distinguishing cell populations that have been tagged with different fluorescent proteins or dyes. 3....progenitor cell migrating to the epithelium). At this stage, only cells from the immune system show wide scale...like EGFP Yellow-orange fluorescent proteins like TagRFP, tdTomato, DsRed, the mKate series, or tdKatushka2...permanent labelling of cells using cre-lox is advantageous when compared to photoactivable or photoconvertible...during the development and disintegration of mycobacterial granulomas." Immunity 28.2 (2008): 271-284. ...presentation and T cell effector function in mycobacterial granulomas." Immunity 34.5 (2011): 807-819. ...

...perfect empty backbone, made sure to add the right tags to your gene, and may have even put a fluorescent...subunit S12 of 30S ribosome Confers resistance to streptomycin Tn10   Confers resistance to tetracycline ...galK rpsL (StrR) endA1 nupG fhuA::IS2 DB3.1 Streptomycin  HB101 derivative. For propagating plasmids ...lacY1 proA2 rpsL20(Smr) xyl5 Δleu mtl1 DH10B Streptomycin MC1061 derivative. General cloning and storage...lacZYA-argF)U169, hsdR17(rK- mK+), λ– HB101 Streptomycin Hybrid of E. coli K12 and E. coli B (mostly ...proAB+ lacIq lacZΔM15] hsdR17(rK-mK+) JM110  Streptomycin  For storing plasmids that should not be Dam...proAB+ lacIq lacZΔM15] hsdR17(rK-mK+)  MC1061 Streptomycin Parent of DH10B/TOP10 and derived strains, common...

...Photoactivatable GFP tagging cassettes for protein‐tracking studies in the budding yeast Saccharomyces cerevisiae...FPs) offer scientists a simple yet powerful way to tag cellular proteins and investigate protein localization...fusion, etc). For example, by engineering PA-GFP tagged to a mitochondrial matrix protein (mito-PAGFP) ...an FP-chimeric protein may be at any one of many stages of synthesis and degradation. For this reason it...PA-FPs by performing several rounds of random mutagenesis screening for enhanced RFP variants. PA-mCherry...negative” photoswitching. In 2008, scientists used mutagenesis screens to find a version of Dronpa that doesn't...

...promoters, terminators, solubility and purification tags, connectors, cDNAs, and more to find the perfect...constitutive and inducible promoters, linkers, and tags makes EcoFlex a great option for those interested... a larger library of parts but don’t need to add tags to your proteins, then the CIDAR Kit is an excellent...Toolkit for Extensive and Flexible Engineering of Saccharomyces Cerevisiae.” ACS Synthetic Biology, vol. 12,...

... al. shows just that - using MMEJ to insert FLAG tags into the genome of the pathogenic fungus Aspergillus...of press. However, this process accounts for a percentage of the mutations seen with TALENs and CRISPR.... between the microhomologies.MMEJ has certain advantages over HDR. Some species don’t have good HDR systems...- and locus-specific gRNAs Step 4: Select for puromycin resistant cells Step 5: PCR amplify and sequence...cassette into the FBL locus. Upon sequencing of puromycin resistant clones, they found that 80% and 50% ...follows. For increased versatility, it would be advantageous to adapt PITCh to insert genes into AAVS1, the...using the nuclease Cpf1. Since Cpf1 cuts in a staggered pattern, it is thought to be ideal for HDR-independent...

...chapters on genome engineering, cloning, and plasmid tags, addtional content added to the "Plasmids that Glow...resistant plasmid, pSC101, and a newly developed kanamycin resistant plasmid, pSC102, with EcoRI and selected...

...uracil DNA glycosylase inhibitor and degradation tag on the fusion protein to limit the activity of the...other advantages including shorter plasmid curing time. pCRISPomyces for editing in Streptomyces  Streptomyces...created two “pCRISPomyces” systems for use in Streptomyces (Cobb et al., 2015). pCRISPomyces-1 includes ...array, while pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays...for transcriptional silencing Mycobacterium tuberculosis and Mycobacterium smegmatis. In addition to their...Addgene depositor Luciano Marraffini’s lab took advantage of this lethality to design the first synthetic...carries a spacer targeting the gene of interest and kanamycin resistance E. coli carrying the phage recombineering...

...the concentration of inducer or adding an affinity tag such as GST may help with solubility issues. We ...toxic or insoluble proteins Origami2 (DE3)** Streptomycin and Tetracycline Contains highly active thioredoxin... General protein expression  m15 pREP4*, ** Kanamycin (pREP4) Cis-repression of the E. coli T5 promoter...

...elements – promoters, origins of replication, protein tags, and antibiotic resistance markers (just to name...recombination at the loxP recognition sites. A key advantage of the system is that Cre acts independently of...in certain cell or tissue types, during certain stages of development, or by making the Cre inducible ...recombination event in a single system. The Saccharomyces cerevisiae FLP-FRT recombination system is another...

...the the developing zebrafish pancreas. They first tagged these cells using plasmids. Then using HOTcre, ...researchers: the mouse (Mus musculus). Mice have many advantages as a mammalian model organism for scientists ...You can find these plasmids at Addgene. Another advantage of mice as model organisms is that they are genetically...in the fly (Pandey and Nichols, 2011). A huge advantage of using the fruit fly is the array of genetic... of the fly (Pfeiffer et al., 2010). Yeast (Saccharomyces cerevisiae) Yeast, one of the simplest eukaryotic...scientists can easily observe early developmental stages which can be difficult in other vertebrate species...Craig Mello a Nobel Prize in 2006. One of  major advantages of C. elegans is that they are transparent throughout...

...Post About Yeast Vectors Read our Blog Post About Tagging Yeast Genes ...Two hybrid systems were developed in Saccharomyces cerevisiae in 1989 and are still used extensively ...activator (1a) or a repressor (1b). Scientists took advantage of these discoveries to create a new system for...subset based on a cellular compartment or growth stage). A prey library can either be applied to the bait...

...Addgene is certainly going to have to find ways to tag and curate reagents from these emerging organisms...So, think beyond Drosophila melanogaster or Saccharomyces cerevisiae (both fine research organisms) -  ...

...Slow transformation   Spectinomycin/streptomycin Spectinomycin and streptomycin both inhibit protein synthesis.... Pros: Stable (spectinomycin). Cons: Cost (spectinomycin) or stability (streptomycin), doesn’t work for... for transformations. Con: Expensive.   Kanamycin Kanamycin, or kan, is an aminoglycoside which inhibits...Like carb, spectinomycin is the more stable and expensive antibiotic compared to streptomycin. Spectinomycin...cleavage in human cells. Mutagenesis, 20(2), 111–114. https://doi.org/10.1093/mutage/gei016I Resources on ...Spectinomycin/streptomycin can be successfully used for plant and bacterial culture, but some types of bacteria...bacteria.   Zeocin  Zeocin is a member of the bleomycin family of antibiotics, which induce cell death...

... ug/mL Bleomycin glycopeptide Induces DNA breaks Bactericidal 5-100 ug/mL Erythromycin macrolide...Additionally, the presence of a plasmid is disadvantageous from the bacterium's perspective – a plasmid-containing...Class Mode of Action*   Working Concentration** Kanamycin aminoglycoside Binds 30S ribosomal subunit; ...mis-translation Bactericidal 50-100 ug/mL Spectinomycin aminoglycoside Binds 30S ribosomal subunit;...protein synthesis Bactericidal 7.5-50 ug/mL Streptomycin aminoglycoside Inhibits initiation of protein...

...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...

...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...

...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...