...will be necessary to improve its multiplexing capacity. Multiplexing is key due to the natural redundancy...graphic below - including some simple comparisons. The easy multiplexing afforded by PTGs will improve...knockout, or multiple gRNAs may be used to target genes with overlapping activity. Multiple gRNAs can also... Addgene Other Techniques for gRNA Multiplexing Multiplexed and programmable regulation of gene networks...;  to observe a phenotype, the modification of multiple genes is often necessary. Guide RNAs (gRNAs) are...editing efficiency. One previous strategy for multiplexing gRNAs used the RNA nuclease Csy4 from Pseudomonas... test the application of this system to gRNA multiplexing, Xie et al. constructed PTGs containing 8 gRNAs...

...see their name tag if you are one of those people. Meet people in sessions by sitting next to someone and...conferences. Conferences provide the opportunity to meet people who are interested in the same things you are on...will meet a potential collaborator Some of these people you meet will review your papers or grants in the...the future, and don’t be surprised to know that people think more favorably of scientists they know personally...depend on your national recognition and the more people you know, the larger your profile in the community...opportunities but can be overwhelming as far as meeting new people. At large conferences, look for smaller events ...media to let others know you will attend. Meet some people online to follow up with in person. Email lab members...

...the SARS-CoV-2 Spike protein and you will find examples of each in Addgene's collection: HIV-based lentiviral...and Packaging Plasmids Reporter Plasmids A few examples of reporter plasmids that can be used for measuring...expressing mCherry and firefly luciferase. pLentiEGFPdestablized - EFS-EGFPd2PEST-2A-MCS-Hygro - Lentiviral...

...Bacterial Expression mApple 568 592 37 6.5 30 min Monomer mApple-N1 - Mammalian Expression mApple-C1 - Mammalian...Mammalian Expression mApple-pBAD - Bacterial Expression mScarlet-I3 568 592 68 4.2 2 min Monomer pmScarlet-...

...Ralstonia solanacearum species complex (Rssc) gene knockout and complementation constructs - Lahaye Lab Vaccinia... in genome editing of bacteria. CRISPathBrick Multiplex Plasmid : CRISPR expression in E. coli - Koffas...

...IBEX is a highly multiplexed tissue imaging method that uses iterative staining and chemical bleaching...Radtke et al., 2020, 2022). I do mean highly multiplexed: you can image over 65 different parameters using...community and the database. They particularly encourage people to upload and share their negative data. After ...an invaluable resource for anyone performing multiplexed imaging, not just IBEX. The IBEX Imaging Community...., & Germain, R. N. (2020). IBEX: A versatile multiplex optical imaging approach for deep phenotyping ...phenotyping and spatial analysis of cells in complex tissues. Proceedings of the National Academy of Sciences, 117...resources on the Addgene blog Antibodies 101: Multiplex Immunofluorescence Negative Can Be Positive: Open...

... in proximity by the looping of chromatin, the complex of DNA and proteins that forms chromosomes. These... of the CAPTURE method. After protein and DNA complexes are cross-linked, DNA is digested into smaller...and also makes it hard to compare results across multiple targets because different CAPTURE cell lines will...and 2) gRNAs Number of loci targeted One Many Sample type(s) Cell lines Cell lines, primary cells ...lab presented three ways to use CAPTURE 2.0: 1. Multiplex capture of chromatin interactions The Xu lab performed...experiment to show that CAPTURE 2.0 could detect multiple chromatin interactions at once. Chromatin interactions...Long-range chromatin interactions identified with the multiplexed CAPTURE 2.0 largely replicated interactions previously...

...replicate.   How can I prevent recombination in my samples? Plasmid recombination can affect viral vectors...vectors (LTRs and ITRs), plasmids with multiple polyA sites, and other plasmids that contain repeating elements...stable E. coli reduce the rate of recombination in samples. For a more detailed comparison of these cloning...following protocols for low copy plasmids. For example, using a longer growth period (up to 24 hr), reducing...rich media can improve growth and DNA yield. Test multiple, small colonies After streaking LB agar plates...cultures contain the recombined vector backbone. Test multiple colonies before saving a glycerol stock to ensure... easiest and clearest ways to determine if your sample has recombined. Choose a restriction enzyme that...

...ensure that samples are easy to locate in freezers that may be shared with multiple people or even multiple.... Some samples are submitted as bacterial colonies in petri dishes, but close to 80% of samples are received...of the samples that come through our doors. It would be a nightmare if we didn’t have a simple way to ...every sample can be tracked easily at each step in the storage process. After the original sample is barcoded...scanned to verify that it is the correct sample. Tracking samples - it's all about location, location, location...these samples have the 2D barcodes mentioned above. These barcodes identify what type of sample is contained...allow us to store multiple types of tube for each plasmid we have at Addgene. Each sample at Addgene has ...

... wide array of genetic functions in cells. For example, if there is a protein thought to be associated..., are suited for specific research goals . For example, adeno-associated virus (AAV) is typically preferred...depending on the insert type and envelope used. Please consult with your institution’s Biosafety Committee...

...affected the pH of our viral samples. Read on to learn how aqueous samples might be affected by dry ice...Measuring the pH of our samples: What we did To measure the pH of our samples, we used a solution of cell...when the samples were frozen. This was clear by the gradual change in pH visible at the sample interface...this, we stored samples on dry ice for 72 hours and then vented the tubes by completely removing the caps...at -80°C. For samples that are shipped on dry ice, this can affect the pH of the sample, which may have...have some effect on the contents of the sample. For example, one study found that dry ice storage affected...describing how short term storage on dry ice can shift sample pH, we wondered whether or not the dry ice we use...

... one simple experiment. PCA is an in vivo technique where two proteins are fused to complementary reporter...neighboring proteins. Thus to capture protein complexes, scientists can simply express a bait protein ...suboptimal folding or stability. Thus they conducted multiple rounds of directed evolution until they identified...interactions Split-BioID combines a protein fragment complementation (PCA) technique with proximity labelling to...labeling and purifying additional proteins from a complex of interest. For more information, read the Split-BioID...detect RNA-protein interactions that can regulate multiple biological functions. RapID (RNA-protein interaction... and Paul A. Khavari. "Research Techniques Made Simple: Emerging Methods to Elucidate Protein Interactions...

... have no fear - I’m here to introduce you to a simpler, yet more effective, CRISPR-based epigenetic approach...machinery.   Well, I’ve got some good news: complete DNA demethylation can be achieved by dCas9 alone...Sapozhnikov and Szyf 2021).    Figure 3: A simpler approach - dCas9 blocks DNMT1 methylation, leaving...too.  High dCas9 and gRNA expression are key for complete DNA demethylation – tight binding is crucial for...blasticidin or puromycin resistance genes.  It’s that simple! Good luck and happy editing!    Thank you to...

...Novophage to Sample 6. To learn more about Novophage's successful transformation into Sample 6 and Dr. ... in food products prior to Sample 6 16:27 - 18:02 : Benefits of the Sample 6 technology and how It works...- 19:56 : Why Sample 6's first test was for Lysteria 19:56 - 24:41 : Who is using Sample 6 and why? 24...Resources Visit the Sample 6 website Check out Dr. Koeris' Website, biotechstart.org Read Sample 6's Paper on...for use in a biotech startup 12:16 - 15:15 : Why Sample 6 began to develop tests for food products as opposed...Directors. We're excited to watch both Addgene and Sample 6 grow with his help! Special thanks to our sound... sample6...

...transcriptional activation domain from HSV (herpes simplex virus). In the absence of tetracycline, the tetR...Puromycin was replaced with Blasticidin selection. Please NOTE, this vector contains an insert (MAT2A) which... Plasmid Description Element On or off PI 26429 pLenti CMV rtTA3 Blast (w756-1) Lentiviral reverse tetracycline-controlled...

...construction system for multiplex genome engineering. Image from Sakuma at al. (2015). Multiplex genome engineering... construction system for CRISPR/Cas9-mediated multiplex genome engineering is now available thanks to ...new tool in Scientific Reports in June. “The multiplexity is one of the most advantageous properties of...established way of making an all-in-one vector for multiplex genome engineering.” Now, they’ve filled that...Cas9-nickase vectors work successfully to target multiple sites in cultured human cells. While it has not...be applied to efficient viral vector-mediated multiplex genome engineering. The system now provides an...an efficient targeting strategy for multiplex editing of genomes. It will also allow simultaneous activation...

... to implement in practice because FRET depends on a number of hard-to-achieve factors. For example, FRET...Bimolecular Fluorescence Complementation (BiFC). What is bimolecular fluorescence complementation? Bimolecular fluorescence... TK (2006) Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the...often easier said than done. Fortunately, a complementary method exists for visualizing protein-protein... fluorescence complementation is a method for detecting protein-protein interactions that uses split fluorescent...fragments that individually lack fluorescence. When complementary FP fragments are fused to two hypothesized protein...interaction partners (PIPs), the FP fragments form the complete FP because the PIPs they’re tethered to bring ...

...Plasmids are available for all the items listed below. Please let us know (Link opens in a new window) what you...you would like to see available in viral format. Please note this does not guarantee viral service, but...pENN.AAV.CB7.CI.TurboRFP.WPRE.RBG James M. Wilson Please contact [email protected] with any questions. Return...

...Rabies Virus-Based Serotype Serotype Testing Samples Small samples of control AAV vectors for trial purposes...

...plasmids for neuronal excitation and inhibition. For example, search for "ChR mCherry", "Cre inhibition", "Boyden...to control a protein or cellular activity. For example, search "Cdc42", "transcription", "Hahn", or other...