...1: 293T cells were transfected with the GFP-expression plasmid pRosetta using μg total DNA to μg PEI ratios... 0.72 pmol Transfer Plasmid* 1.64 pmol OptiPro SFM to total volume 500 μL *Plasmid concentrations and ... each transfer plasmid. Pro-Tip Endotoxins can inhibit transfection; therefore, plasmid DNA purification...endotoxin removal step. For high quality plasmid DNA, the plasmids should also be propagated in an endonuclease... cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA 27.8 μg), this would be...~20 h. Prepare a mixture of the 3 transfection plasmids into OptiPro SFM: Reagent Amount per 10 cm dish...is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 ...

...therefore, plasmid DNA purification should include an endotoxin removal step. For high quality plasmid DNA, ...protocol to transfect mammalian cells with your plasmid of interest. Protocols...polyethylenimine. Transfections allow for transient expression of a gene of interest in a target cell line and..., the plasmid should also be propagated in an endonuclease negative E. coli strain such as NEB stable....is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1–2 ...determine what ratio gives the highest percentage of GFP positive cells. Refer to the table below for a possible...the cells 24–48 h before checking for protein expression. Sample Data Legend: Lenti-X 293T cells were ...

...Cell marking/selection: Expression constructs like plasmids often include GFP as a marker to help identify...scientists to begin to study GFP and deduce its biochemical properties. Now GFP and its fluorescent derivatives...employ GFP for a wide number of functions, including: tagging genes for elucidating their expression or localization...biology. Scientists can easily utilize GFP-containing plasmids as a means to many functional ends. We’...taken up the plasmid. This can serve as an alternative to antibiotic selection. Plasmids of this type ...signal. Thus, FACS can be used to separate cells expressing GFP from cells that are not. Developmental/transgenic...being tested. Find FRET plasmids here. Split EGFP: An alternative to FRET, split EGFP has also been used to...

...control plasmids in new serotypes, as well as a new Cre dependent GFP control plasmid for expression in forebrain...Lentiviral preps for expressing SARS-CoV-2 proteins ChR2 for targeted expression DREADDs GFP dependent Cre and...preps SARS-CoV-2 protein expression The Krogan lab quickly shared plasmids expressing SARS-CoV-2 proteins ...for targeted expression. Plasmid 75470 is now available in AAV1 for Flp-dependent expression of ChR2(H134R...restrict gene activation to cells expressing both Cre and GFP. The GFP dependent FLPo, Flp-DOG (75469), ...2xStrep-IRES-Puro: Expression of the SARS-CoV-2 ORF3a Lentiviral preps for expressing the E protein (pLVX-EF1alpha-SARS-CoV...reporter. We now also offer AAVrg for plasmid 83898, which targets expression of ChR2 to forebrain GAB-ergic ...

...GBPs/GFP. The new system, CRE-DOG (Cre Dependent On GFP), is activated by GFP and derivatives GFP and ...reporter expression was induced in ~76% of GFP+ cells, and 100% of DsRed+ cells also expressed GFP. Using...mouse lines expressing GFP in specific cell types to do more than just label cells. If GFP could be co-opted...to bind GFP. The idea of using GFP as a scaffold suddenly seemed very realistic. Envisioning GFP as a substrate...electroporated T-DDOGs, GFP, and a reporter construct into the mouse retina. GFP expression robustly activated...whose expression was absent without electroporated GFP. They then tested T-DDOGs with two mouse GFP reporter...presence of GFP. These systems, known as T-DDOG and Cre-DOG, respectively, repurpose popular GFP reporter...

...released Anti-GFP [N86/8R] as another option for targeting GFP. But why might you need a second GFP antibody...western blot for mEGFP-HRas (from Plasmid 18662). See the Applications section on the Anti-GFP [N86/8R] page...of the new plasmids, antibodies, and viral preps in the repository through our hot plasmids articles. ...region-specific, inducible expression.   Find heat-inducible lentiviral plasmids here!   Wu Y, et. al. Control...PMCID: PMC9015817.   Meet anti-GFP [N86/8R]! by: Ashley Waldron Anti-GFP [N86/38.1R] was among the first...effects caused by T cell migration.  CAR expression (or the eGFP proxy for CAR) is elevated at stable levels... missed something smokin'? You can pitch a Hot Plasmids section (antibodies and viral preps welcome!) ...

... recruits GFP fused to the cognate scFV antibody, which is expressed from a separate plasmid. This system...al. observed some GFP aggregation, which they reduced by using superfolder GFP (sfGFP) with the small solubility...scaffold fully occupied with GFP has a molecular weight of 1,400 kDa vs 24 kDa for GFP alone. Although traditional...nomenclature throughout the rest of this blog post, GFP refers to sfGFP-GB1.   Tanenbaum et al. examined the power...protein is localized, and how its localization and expression may change across various conditions. However...perfect. For example, If your fusion protein is expressed at low levels, you have to increase your imaging.... then tested their hypothesis that the lower expression levels of SunTag constructs would avoid negative...

...thought I could see GFP expression with a fluorescence microscope, but the GFP expression was so low (even... against GFP expression, but I didn’t. 2. In the original publication they see GFP expression in about...from BRAF-edited and GFP-edited amplification products (BRAF-GFP duplex) and from GFP-edited amplification...cells to express Cas9. In these particular experiments, we generated A549 cells stably expressing Cas9 via...wildly variable Cas9 expression (Figure 1). This is a problem because Cas9 expression affects growth rate...variable Cas9-expression was across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal...Checking Cas9 expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but...

...recombinase. If GFP is under control of an endogenous promoter, you can use expression GFP to track cells...tag an endogenous protein with GFP, as seen in blue flame plasmid OCT4-eGFP-PGK-Puro from the Jaenisch lab...knockout a given gene and insert GFP into the genome, we’d create a plasmid similar to the one shown below...is disrupted (knocked out) but the inserted GFP is expressed (knocked in). As seen in the above example...designed to insert GFP into a given locus.   Conditional knockouts The methods and plasmids described in...on Addgene.org Find Mammalian Expression Vectors Find Cre-Lox Plasmids Learn More about CRISPR  ...DNA designed in the lab. Specially constructed plasmids can be used to replace genes in yeast, mice, or...

...vectors and an envelope expressing plasmid, such as our second most requested plasmid of all time, pMD2.G....applications. This plasmid was the top requested plasmid of 2017. Find pMD2.G. pSpCas9(BB)-2A-GFP (PX458) - This... the envelope expressing plasmid pMD2.G. Find pMDLg/pRPE. scramble shRNA - This plasmid from David Sabatini...BB)-2A-GFP (PX461) expresses both a Cas9 protein and a gRNA. But instead of Cas9, this plasmid expresses...generation lentiviral packaging plasmid from the Didier Trono lab that expresses the HIV gag, pol, rev, and...Didier Trono lab is pMD2.G, a VSV-G envelope expressing plasmid. Envelope proteins determine which types ... This plasmid from the Zhang lab expresses Cas9 from Streptococcus pyogenes with an invariant sgRNA scaffold...

...using the supplied donor plasmid will express GFP. If there’s GFP expression, presumably the endogenous...control RFP expression plasmid.  With this set up, all successfully transfected cells will express RFP while...RFP to GFP expressing cells provides a real-time read out of Cas9 activity.  By using the RFP:GFP ratio...  A donor plasmid that contains a puromycin resistance gene linked to an in-frame H2B-GFP. This visual...gene NFE2L2, the Madison lab found that H2B-GFP expressing cells exhibited a 2-4 fold increase in the ...integrated GFP reporter leaving you with just your desired genomic edit.  Find SRIRACCHA plasmids at Addgene...in the genome) and an out-of-frame H2B (histone)-GFP reporter into the genome of mammalian cells.  ...

...capsid (cap) expressing plasmids is generated. Diversity is introduced into the cap plasmid library by ... comparison, capsids were packed with a GFP transgene and GFP intensity and transduction efficiency of...and the level of transduction, measured via GFP expression, was determined 3 weeks later. Table was derived...are easy to manipulate, 2) they have long-term expression; and 3) they have limited toxicity. However, ...Caltech stepped up to the challenge. Find the PHP Plasmids Here! Their Solution: Use the CREATE method to...region of the cap gene. In this special CREATE cap plasmid, loxP sites flank the downstream polyA sequence...

...determined by comparing cells expressing only eGFP expression to cells expressing eGFP and mCherry. ACE was used...mCherry and a T2A site and expressed in cells. mCherry is constitutively expressed to identify successfully...efficiency of plasmid delivery to a cell. In contrast, TREE provides a read out for plasmid delivery and...engineered BFP variant that converts to GFP after a base editing event. GFP positive cells can be sorted and ... their hypothesis with GFP fluorescence. To do this they generated a silent GFP construct, that contains...the histidine to a tyrosine generating a GFP variant (GFPY66) that has a shifted emission spectra. Thus...that can be repaired by NHEJ restoring mCherry expression. Cells can then be sorted using Fluorescence-...

...CatCH-GFP reporter. Expression of GFP will only result in cells where Cre-mCherry is also expressed. If ... fused to a fluorescent GFP marker (CatCH-GFP). The lab deposited AAV plasmids containing the active promoters...to test for cell type-specific expression of GFP. Half (113) expressed in retinal cells four weeks post-injection...Cre-mCherry expresses in photoreceptors and horizontal cells, and Cre-dependent-GFP expresses in horizontal...More than 90% of expression occurred in one type of cell More than 50% of expression occurred in one type...of targeted expression. Surprisingly, <1% of synthetic promoters replicated the expression specificity...restrict expression to a single cell type based on the overlap in cell types both promoters expressed in. ...

..., “sender” cells express GFP on the surface and “receiver” cells express an anti-GFP nanobody on the cell...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...pairs. Because the anti-GFP nanobody cross-reacts with BFP, sender cells expressing surface BFP generated...response 1 (TIR1) expressed under a desired promoter with a specific spatiotemporal expression pattern and ...highlights from recent CRISPR plasmids. To find all of the CRISPR plasmids available from Addgene, head...head over to our CRISPR Plasmids and Resources page.  Plasmids for delivering CRISPRi components by a single...

...can then be co-expressed in essentially any transgenic zebrafish line expressing a GFP-tagged version ...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need... proximity to the GFP-tagged protein (albeit slightly offset due to the nanobody-GFP interaction). The...display increased neuronal expression in the brain while having decreased expression in the liver after systemic...only AAV.CAP-B10 causes brain wide expression, with no liver expression, after retro-orbital sinus vein ...need. If you'd ever like to write about a recent plasmid deposit please sign up here. Here's what you'...

...dim GFPs The availability of the three dimensional structure of GFP, its brighter mutant S65T-GFP, and...Cre-based Brainbow-expressing cells. Autobow plasmids are all-in-one versions which express self-excising ... host cells in order to optimize expression. The expression plasmids for a given species are based on ... Hot Plasmid list. Other popular plasmids & blog posts @Addgene: The 10 Most Distributed Plasmid Technologies...inducible inactivation. Find the Robinson plasmids or the Royle plasmids and get to work! Robinson et al...strain in which Cre expression causes the induction of both DsRed fluorescence and GFP shRNA. Mice carrying...carrying the R26DsRedR; CRUSH-GFP; and Nestin-Cre alleles showed efficient GFP knockdown in eyes and clonogenic...

...Chemogenetics Plasmid Serotype Name 119472  AAV5  AV SYN PSAM4 GlyR IRES EGFP   Biosensors Plasmid Serotype...AAV encoding GFP. Check out our inventory of serotype testing AAV New control AAVs Plasmid Serotype Name...populations. We now offer a Flp-expressing AAV, as well as a Cre-dependent Flp-expressing AAV. See our recombinase...optogenetics inhibitors Plasmid Serotype Name 99039  AAVrg pAAV-CamKII-ArchT-GFP (PV2527) 105679  AAV5...Controls Plasmid Serotype Name 37825  AAVrg (20 µL size serotype testing) pAAV-CAG-GFP 50465 AAV1... Name 105530  AAV1 pAAV.CMV.PI.EGFP.WPRE.bGH 37825  AAV1 pAAV-CAG-GFP 59462  AAV2 pAAV-CAG-tdTomato... testing) pAAV-CAG-GFP 50465 AAV8, 9 (20 µL size serotype testing) AAV-hSyn-EGFP Biosensor AAV The...

...limitations of GFP photoconversion One concern with photoconverting GFP is the expression level necessary...process worked well with the common GFP variants EGFP, mGFP4-T, and S65T GFP, and no red color was observed...exciting GFP-labeled plant cells at 405 nm instead of 488 nm, Sattarzadeh et al. noticed that GFP had turned...cytosol-targeted GFP. White circles represent the irradiated area. Photoconverted GFP spreads through ...intensity increases with GFP concentration and saturates around 30 uM. For low-abundance GFP fusions, a photoconversion...repurpose GFP is great news for microscopy enthusiasts! Addgene has empty backbones containing GFP variants... Find plasmids from this publication at Addgene.   Addgene’s Fluorescent Protein Plasmids and Resources...

...cloning and expressing your gene of interest with GFP expression.  Cloning and expressing RNAi pMKO.1 ...packaging plasmids as well as transfer plasmids. This concept ensures safe handling and expression of these...gamma-Retroviral Transfer Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) Retroviral vector...technology CAG-GFP-IRES-CRE (Plasmid #48201) This retroviral vector from the Gage lab encodes GFP and Cre-recombinase...separating viral genes and transgene expression across several plasmids. The most commonly used viral vector...gamma-retroviral packaging plasmids are not interchangeable. General envelope plasmids, such as VSV-G, may be.... CRISPR technology pdCas9-humanized (Plasmid #44246) Expression of a catalytically inactive (dead), human...