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We narrowed to 573 results for: BASE

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  1. 10 Great Guest Posts We're Thankful For

    Type
    Blog Post
    Published
    Nov. 18, 2016, 3 p.m.
    ...your next experiment. Nucleic acid based machines Recombinase-based State Machines Enable Order-dependent...Roquet from the Lu Lab at MIT developed plasmid based systems that report the order in which specific ...received by a cell. These so-called recombinase-based state machines could potentially be used to report... Read this post to learn more about recombinase-based state machines and how they function. Adapting Toehold... Toehold Switches to Detect Zika Virus The RNA-based toehold switches described in this post are engineered...
  2. Choosing the B(right)est Fluorescent Protein: Aggregation Tendency

    Type
    Blog Post
    Published
    June 15, 2017, 2:30 p.m.
    ...under non-denaturing conditions separate proteins based on size. Whether higher molecular weight complexes...tendency. Another technique that separates molecules based on size is ultracentrifugation. Sedimentation equilibrium...protein forms dimers. Finally, spectroscopic methods (based on fluorescence polarization) can be used to detect...EGFP. Constantini et al, (2012) developed a cell-based assay that does a better job of detecting homodimerization...whereas it was previously concluded to be monomeric based on gel filtration (Chudakov et al, 2007). We have... and negative controls for proper ER structure. Based on these data, we conclude that mKOk has dimerization...Amsterdam) where she develops genetically encoded FRET based biosensors.     Additional Resources on the Addgene...
  3. Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

    Type
    Blog Post
    Published
    Sept. 26, 2024, 1:15 p.m.
    ...insert genes into plasmid backbones, base pairing between these ~4 base overhangs isn’t strong enough to ...recombination, it also doesn’t require a ligation step. Based on the robust system of homologous recombination...insert are (separately) digested to create 10–12 base “chewed back” overhangs, they can be annealed, forming...preventing T4 from chewing back the entire 10–12 bases. As such, the use of LIC is often limited to specially...overhangs must be accessible to allow for complementary base pairing, so SLIC can’t be used if the overhangs ...compared to Gibson assembly, a cloning method also based on homologous recombination. The higher temperature...
  4. Hot Plasmids - February 2022

    Type
    Blog Post
    Published
    Feb. 22, 2022, 2:15 p.m.
    ... in AAV. A dCasMini-mediated adenine base editor allows for base editing, while gene editing can be done...perturbation by: Gabrielle Clouse While many CRISPR-based genetic screening methods have primarily used Cas9...genetic screening toolkit by optimizing a Cas12a-based method for screens. Cas12a’s ability to perform ...multiple genes in parallel. Lastly, this Cas12a-based screening method was used to create a genome-wide... Rachel Leeson This simple, inexpensive plasmid-based ladder system is a cost-effective alternative to...efficacy to dCas12a systems in both gene activation and base editing applications.  Figure 4: Overview...
  5. Cpf1 Update: Comparison to Cas9 and NgAgo

    Type
    Blog Post
    Published
    July 14, 2016, 2:30 p.m.
    ...Potential advantages of Cpf1 Type II CRISPR systems based on Cas9 were thought to be the simplest CRISPR systems...fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. Cpf1’s staggered cleavage pattern...potential to eclipse any of the CRISPR nucleases in AAV-based applications. Get More Cpf1 Background Here THE ... Figure 1: Effects of (A) double and (B) single base pair gRNA-target mismatches on modification by AsCpf1...with single and double mismatches throughout the 23-base sequence. Double mismatches ablated Cpf1 activity...were present in the 3’ end of the target sequence (bases 19-23). Cpf1 is also sensitive to single mismatches...et al. saw no decrease in Cpf1 activity with 4-6 base deletions at the 3’ end of the target sequence. ...
  6. Cellosaurus: A Cell Line Information Database

    Type
    Blog Post
    Published
    Feb. 21, 2023, 2:15 p.m.
    ...score match. This tool’s algorithm can be adjusted based on query, reference, and shared allele search preferences...called Cellosaurus, a growing cell line information database describing nearly 145,000 cell lines. Cellosaurus...researcher, Cellosaurus was able to both update their database and sent the updated information to the cell line...
  7. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    Published
    Oct. 27, 2020, 1:15 p.m.
    ...     A new positive selection-based RNAi method Until now, all DNA-based RNAi experiments typically involved...pervasive off-target effects. This conclusion was based upon numerous phenotype discrepancies between gene...considerations present at the first stage of an RNAi-based investigation. Creation of the RNAi trigger requires...considerations, such as the starting material (DNA-based vector or purified RNA) and the initial state of...allows easy creation of long hpRNA with Gateway-based cloning and straightforward positive selection of...occurrence of off-target effects. Nevertheless, amiRNA-based RNAi experiments routinely display highly specific...#137884). Both vectors allow the creation of amiRNAs based upon miRNA precursors of rice and Arabidopsis species...
  8. To Codon Optimize or Not: That is the Question

    Type
    Blog Post
    Published
    Nov. 12, 2020, 2:15 p.m.
    ... created a codon adaptation index (CAI) that is based on the codon usage frequency in a reference of highly...improve gene expression by changing synonymous codons based on an organism's codon bias. The idea is that scientists...can make mutations throughout a gene of interest based on an organisms codon usage bias to increase translational...code (Gustafsson et al., 2004), such as Hoogsten base pairs and wobble pairs.  Figure 2: (A) The...different tRNAs, including those that exhibit wobble base-pairing (a tRNA that can recognize multiple synonymous...Chappell SA, Zhou W, Mauro VP (2005) An mRNA-rRNA base-pairing mechanism for translation initiation in ...-0270(02)00362-x Stadler M, Fire A (2011) Wobble base-pairing slows in vivo translation elongation in ...
  9. CRISPR 101: RNA Editing with Cas13

    Type
    Blog Post
    Published
    July 31, 2020, 12:30 p.m.
    ...a 30 nt guide when C or U base-flips are present across from the target base. However, they saw that in...Cas13 enzymes prefer targets with a given single base protospacer flanking site (PFS) sequence, but orthologs...so they cannot directly edit the genome. A Cas13-based RNA editing system would likely be reversible and...it is sensitive to mismatches in target RNA from bases 12-26 of a 30 nt target sequence. For the editing...targeting work with Cas13, REPAIR is the first CRISPR-based system to enable precise RNA editing. The addition...al. “A cytosine deaminase for programmable single-base RNA editing.” Science 2019 Jul 26;365(6451):382-...RNA Targeting with Cas13a Learn about CRISPR DNA Base Editing Resources on Addgene.org Browse All CRISPR...
  10. PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs

    Type
    Blog Post
    Published
    June 25, 2024, 1:15 p.m.
    ... pegRNAs. Chromatin-based prime editing efficiency prediction While sequence-based prediction models from...building a chromatin-based prediction tool (ePRIDICT). In summary, machine learning-based prime editing ...way to predict the efficiencies of pegRNA designs based on patterns observed from high-throughput experiments...(used for PE3 and PE5 prime editing strategies) based on a DeepSpCas9 score ranking (Kim et al., 2019)... predicts how well a genomic site can be edited based on its chromatin environment. After selecting the...activity prediction by DeepSpCas9, a deep learning–based model with high generalization performance. Science...
  11. Cpf1: A New Tool for CRISPR Genome Editing

    Type
    Blog Post
    Published
    Oct. 14, 2015, 2:30 p.m.
    ...Class 2 CRISPR systems, including the type II Cas9-based system, require a single-component nuclease to mediate...fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. With this information, they turned...advantages of Cpf1 over Cas9 Type II CRISPR systems based on Cas9 were thought to be the simplest CRISPR systems...with single and double mismatches throughout the 23-base sequence. Double mismatches ablated Cpf1 activity...were present in the 3’ end of the target sequence (bases 19-23). Cpf1 is also sensitive to single mismatches...et al. saw no decrease in Cpf1 activity with 4-6 base deletions at the 3’ end of the target sequence. ... Figure 2: Effects of (A) double and (B) single base pair gRNA-target mismatches on modification by AsCpf1...
  12. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    Published
    June 24, 2020, 5:45 p.m.
    ...previously uncharacterized enhancers. The popular TALEN-based LITE system, which uses light to regulate transcription...deacetylases. CRISPR and epigenetics As with many TALEN-based technologies, the advent of CRISPR has made targeting...strand breaks. Below you'll find some of the CRISPR-based epigenetic modifiers available from Addgene. For...Albert Cheng’s lab developed Casilio-ME, which is based on their Casilio system. This system allows for ...Epigenetic modification is certainly not the only CRISPR-based technology designed to alter gene expression. Fusing...cloning platforms for transgene expression and dCas9-based epigenome editing. Nucleic Acids Research 47:e23...Gersbach CA (2015) Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters...
  13. Identifying Sequence Elements with SnapGene's Feature Database

    Type
    Blog Post
    Published
    Dec. 21, 2017, 2:06 p.m.
    .... Development of this tool required creating a database of common features, and devising rules for identifying...when the match is imperfect. SnapGene's feature database The source of common features was our collection...issue is addressed by adding more variants to the database. Another limitation is that by tolerating mismatches.... To prevent misidentification, we augment the database with new fluorescent protein variants. The future...features? We plan to continue updating the feature database. Powering the plasmid maps on Addgene.org with... which common features need to be added to the database – we will work to fill any common annotation gaps...SnapGene supports “Collections”, which are shareable databases of DNA and protein sequences, the next step is...
  14. Scientific Sharing in the Time of COVID-19: Databases and Resources

    Type
    Blog Post
    Published
    May 12, 2020, 1:15 p.m.
    ...of research into COVID-19. While this program is based in Canada, much of the information is applicable...support this pace of research, new, centralized databases and centers to support the sharing of materials...into COVID-19 (like here at Addgene!), and new databases are supporting their access to useful resources...internationally COVID-19 Resources Canada is a database that provides information about reagents, funding...the future of research. To find other COVID-19 databases and resources, check out our growing Twitter thread...
  15. 15 Hot Plasmids from 2017

    Type
    Blog Post
    Published
    Jan. 9, 2018, 3:02 p.m.
    ...Coselection with Ouabain New CRISPR base editors The popular CRISPR base editing technique uses dCas9/Cas9...specificity of base editing. Kim et al. used natural and engineered Cas9 variants to develop five new base editors...number of available target sites for base editing. For each base editor, they observed editing activity...cytidine deaminase portion of the base editor to create SpCas9 base editors with editing windows as small...off-target effects associated with base editing, Rees et al. created HF-BE3, a base editor containing high fidelity...diseases. The Tol2 Gateway-Compatible Toolbox is based on the original Tol2kit generated by the Chi-Bin...These exciting papers further show the potential of base editing for precise genome modification. Plasmid...
  16. Hot Plasmids Podcast Episode 3: Optogenetics Tools, Improved CRISPR Base Editors, & More!

    Type
    Blog Post
    Published
    Oct. 25, 2017, 1 p.m.
    ...increasing CRISPR editing efficiency, new CRISPR base editors, DIY DNA ladders. You can find additional...Segement on Ouabain Selection  Hot Plasmid 3: CRISPR base editors with enhanced range and specificity. Listen...Listen to to the Segmennt of Improved CRISPR Base Editors Hot Plasmid 4: pPSU Plasmids for generating DNA...
  17. Harnessing TnpB for Genome Editing: A Compact and Versatile Tool for Your Research

    Type
    Blog Post
    Published
    July 29, 2025, 1:15 p.m.
    ... the target site. Additionally, the TAM changes based on the species of origin of the TnpB protein, much...access to regions previously unreachable with Cas-based editors. TnpBs have a familiar targeting mechanism... bound, TnpB cleaves the DNA at a position 15–21 base pairs downstream of the TAM, creating staggered ...Even for advanced genome editing approaches like base editing and prime editing (Molla et al., 2021), ...currently unavailable, you can use TEEP — a tool based on animal data. Step 2: Prepare Oligos Oligo... invite researchers to explore and use our TnpB-based genome editing vectors. If you have specific questions...Qi, Y. (2021). Precise plant genome editing using base editors and prime editors. Nature Plants, 7(9), ...
  18. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    Published
    April 13, 2021, 1:15 p.m.
    ...ex: genome editing), our MUCICAT system which is based on the CRISPR-associated transposase system, INTEGRATE...gene expression is an alternative versus plasmid-based expression which can be unstable and lack precise...application of a multiplex genome editing system based on CRISPR-associated transposition.     References...
  19. What's New in CRISPR - May 2019

    Type
    Blog Post
    Published
    May 28, 2019, 12:49 p.m.
    ... editing (SECURE) base editors Keith Joung’s lab recently found that a cytosine base editor (CBE) with...Covalent tethering of DNA template to Cas9 SECURE base editors Nme2Cas9 CRISPR interference in Candida ...efficiency. Read the paper in Nature Find the SECURE base editors All-in-one AAV delivery of Nme2Cas9 with...
  20. Designer PUF Proteins for Any RNA Target

    Type
    Blog Post
    Published
    June 17, 2014, 7:57 p.m.
    .... “You can think of changing bases to other RNA bases or unnatural bases,” she said. “The possibilities...modular repeats, each of which binds a consecutive base in its RNA target. By changing amino acids, it’s...
  21. Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More

    Type
    Blog Post
    Published
    Aug. 13, 2019, 12:58 p.m.
    ...initially recognized as a useful vertebrate model based on their straightforward husbandry and ability to...developmental biology that arose from zebrafish-based investigations. Genetic resources for zebrafish...important insights in developmental biology. Microscopy-based lineage tracing Over a period of 24 hours, the externally...approaches (Keller et al., 2008). This microscopy-based lineage tracing defined a high-resolution map of...from the random mutagenesis screens. Microscopy-based lineage tracing can help illuminate how signaling...during embryo development. Single cell sequencing-based lineage tracing Recently, gene expression in all...rather than novel, abnormal cell types. Sequencing-based lineage tracing can help elucidate the gene networks...
  22. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas

    Type
    Blog Post
    Published
    May 5, 2020, 1:15 p.m.
    ...overview of the general schematic of CRISPR/Cas based COVID-19 testing methods.   Rapid detection of...2019 novel coronavirus SARS-CoV-2 using a CRISPR based DETECTR assay Sensitivity:10-100 copies of RNA per... al., 2020). Detecting coronavirus using CRISPR-based SHERLOCK (Specific ​H​igh ​s​ensitivity ​E​nzymatic... A scalable, easy-to-deploy, protocol for Cas13-based detection of SARS-CoV-2 genetic material (CREST)...on the target regions thus making it an affinity-based method. Due to the mismatch sensitivity of FnCas9...Wadford DA, Chen JS, Chiu CY (2020) CRISPR–Cas12-based detection of SARS-CoV-2. Nature Biotechnology. https...coronavirus SARS-CoV-2 sequence detection method based on CRISPR-Cas12. Cold Spring Harbor Laboratory. ...
  23. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

    Type
    Blog Post
    Published
    Dec. 15, 2016, 3:57 p.m.
    ...be used for cloning or genome engineering and is based on homologous recombination. It allows for direct...non-selectable sequences, point mutations or other small base pair changes, and the addition of protein tags. ...best for point mutations or changes of only a few base pairs.  dsDNA Substrate dsDNA inserts can be made...the DNA sequence of interest and flank it with 50 base pairs of homology to the targeted insert site (Figure...way is to introduce a C/C mismatch at or within 6 base pairs of the edit site. This is an easy fix, but...the wobble codons, i.e. make changes to the third base pair of the adjacent 4-5 codons that alter the nucleotide... DL. Recombineering: A Homologous Recombination-Based Method of Genetic Engineering. Nature protocols....
  24. Viral Vectors 101: Calcium Sensors

    Type
    Blog Post
    Published
    Aug. 10, 2023, 1 p.m.
    ... the Cameleon-based FRET sensor D1ER, and GCaMPER. Bonus: There are also Cameleon-based FRET sensors available...kinetic parameters. There are three GCaMP types based on their kinetics: GCaMPf (fast) GCaMPm (medium)...wavelength is insensitive. Additional red sensors based on mRuby (jRCaMP1) and mApple (jRGECO1a) are also...reduce these unwanted variables. Twitch is a FRET-based calcium reporter derived from troponin C. It is ...mechanism-guided design of single fluorescent protein-based biosensors. Nature chemical biology. 2021 May;17...
  25. Tips for Titering Your Lentiviral Preps

    Type
    Blog Post
    Published
    March 15, 2016, 2:30 p.m.
    ... of viral particles in a sample and are usually based on the presence of a viral protein, such as p24,...incorporated into a viral particle. Consequently, titers based off of p24 quantification tend to be overestimates...depending on the vector backbone. When using qPCR based methods for viral titration consider using a reverse...by antibiotic-resistance or single cell sorting based on expression of a surface marker, subcloning, expansion...more straightforward approaches such as the FACS-based or colony-forming unit assays, whereas labs that...concentration and titration of pseudotyped HIV-1-based lentiviral vectors."Nature protocols 4.4 (2009):...
  26. Career Coaching for Scientists: Why and Where Do I Find One?

    Type
    Blog Post
    Published
    Jan. 29, 2015, 1:25 p.m.
    ...is another very experienced and successful coach based in New York. Another experienced coach and speaker...you. There is also a group The Dissertation Coach based in Illinois. A few of the coaches in this group ...specialty for Michelle Capes at Adeptify Me. While based in Madison, Wisconsin she also works remotely and...recruiter. Chris Humphrey from Jobs on Toast is based in the United Kingdom and scientists there would...content for PhDs and postdocs posted by Toronto-based Jennifer Polk of From PhD to Life. She runs great...interesting and it is in English and Spanish. She is based in Spain. The bottom line – there are lots of people...
  27. Evolution of Lab Techniques

    Type
    Blog Post
    Published
    June 21, 2016, 2:30 p.m.
    ...fluorescently labeled probes. Colorimetric and luminescence based detection methods are also commonly used. In all... Since the sequencing gel will separate the DNA based on size, the resulting gel should ultimately contain...correspond to every nucleotide in your DNA sequence, based on which ddNTP was added and when. Compiling the...fluorescently labeled ones allowing for fluorometric-based detection. This increased sequencing speed ultimately...world, one up and coming innovation is nanopore-based DNA sequencing which reads the nucleotides of a ...nucleotides (8). Compared to older technologies nanopore-based DNA sequencing machines are relatively small meaning...error rates (10). It is projected that nanopore-based sequencing technologies could one day sequence an...
  28. HA Frankenbody, a New Imaging Tool to Visualize Single Molecules and Nascent Peptides

    Type
    Blog Post
    Published
    Oct. 10, 2019, 12:41 p.m.
    ...and image for long periods of time.  Are antibody based probes a solution? To address these limitations,...these to tag their proteins of interest. Antibody based probes, such as antigen-binding fragments (Fabs)...frankenbody To combat the issues with these antibody based probes the Stasevich lab used an alternative strategy...strategy to develop a genetically encoded antibody based probe that detects the widely used HA epitope in...CDR grafting to stable frameworks and structure-based framework engineering. Methods, 34(2), 184–199. ...
  29. Sequencing Options for CRISPR Genotyping

    Type
    Blog Post
    Published
    Oct. 4, 2016, 2:30 p.m.
    ... types Must be verified with cell-based method IDLV 1% Cell-based Programmable, can detect DSBs in live... genome and pinpoint the location of mismatches based on the sequences of the genome and sgRNA. This is...have disastrous health effects in the clinic. NGS-based methods provide the most complete information profile...al. 2016) and HTGTS (Frock et al. 2015) for cell-based experiments. These strategies can be used in concert...off-target editing events. Two of the most common cell-based methods are genome-wide, unbiased identification... Advantages Disadvantages GUIDE-Seq 0.1% Cell-based  Searches the genome for all DSBs, doesn't require...methods, high background BLESS Not reported Cell-based (in vitro) Can be used on tissue from whole animal...
  30. Fluorescent Proteins 101: Introduction to FRET

    Type
    Blog Post
    Published
    June 27, 2017, 1:03 p.m.
    ...George Abraham, Bobin., et al. "Fluorescent protein based FRET pairs with improved dynamic range for fluorescence...voltage sensors), to name a few.   Find Many FRET-Based Biosensors on Addgene's Biosensor Pages   Recent...Atsushi, et al. "Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin." Nature... Fanjie, et al. "A fluorescence energy transfer‐based mechanical stress sensor for specific proteins in.... Gayrard, Charlène, and Borghi, Nicolas. "FRET-based molecular tension microscopy." Methods 94 (2016)...about Fluorophore Targeting for FRET Studies FRET Based Biosensors Additional Resources on Addgene.org ...
  31. Editor's Choice, August 2016

    Type
    Blog Post
    Published
    Sept. 2, 2016, 1:32 p.m.
    ...system. Site Directed Mutagenesis by PCR & Single Base Editing with CRISPR For my editor’s choice picks...picking two posts that describe methods to make single base edits in DNA. The first, contributed by guest blogger...Scientist Mary Gearing and discusses a newer, CRISPR-based technique to generate C->T or G->A transitions in...Tips for Troubleshooting Viral Transductions Single Base Editing with CRISPR Cas9 Activators: A Practical...
  32. Plasmids 101: Plasmid Incompatibility

    Type
    Blog Post
    Published
    March 31, 2020, 1:15 p.m.
    ... et al., 1998). There are two models for iteron-based plasmid replication control that are not necessarily...synthetic biologist who wants to develop a plasmid-based tool that is easy to use with other plasmids.  How...developed a system of bacterial incompatibility groups based on similarity of replication and partitioning systems...determine how compatible their plasmids will be based on sequence data. However, if these do not provide... antimicrobial resistance. Using novel plasmids based partly on plasmid incompatibility with antibiotic-resistance... R, Rech J, Bouet J-Y (2015) Imaging centromere-based incompatibilities: Insights into the mechanism of...microbial plasmids: classification and identification based on replication and transfer systems and host taxonomy...
  33. CRISPR 101: Validating Your Genome Edit

    Type
    Blog Post
    Published
    Nov. 3, 2022, 12:15 p.m.
    ...least ~200 base pairs of sequence flanking the editing site on either side of the amplicon. Based on the ... A specific insertion or deletion? A knock-in? A base edit? Once you know exactly what you are looking...repair events, or maybe you’re performing a single base pair edit with a prime editor. What next? When generating... you introducing a large insertion, something 20 base pairs or bigger? Then screening by PCR may be the...knock-ins Is your knock-in edit small, with only a few base pairs modified? In these cases, it’s best to do ...Restriction enzyme screening is sensitive to single base pair changes, but the edit must occur at an existing...
  34. Proximity Labeling: A Powerful Tool for Protein Complex Purification and Proteomic Mapping

    Type
    Blog Post
    Published
    Dec. 2, 2019, 2 p.m.
    ...interactions scientists have long used antibody based affinity purification and mass spectrometry approaches...scientists have developed newer methods termed proximity-based labeling to better capture these interactions in..., which can be a challenge for classic antibody based affinity purification/mass spectrometry methods....efficiently label neighboring proteins The first biotin based proximity labelling technique, BioID, was developed...these issues, the Ting lab utilized yeast display-based directed evolution to generate two mutants, the ...interaction detection) uses proximity protein labeling based on BioID to identify RNA-protein interactions (Ramanathan...Trinkle-Mulcahy, L. (2019). Recent advances in proximity-based labeling methods for interactome mapping. F1000Research...
  35. A New Optogenetic Tool Based on AraC Controls Gene Expression with Blue Light

    Type
    Blog Post
    Published
    June 17, 2021, 1:17 p.m.
    ... AraC transcriptional regulator Their system is based on the AraC protein that controls transcription ...pBLADEONLY: This plasmid contains only BLADE and is based on the pTRC99a plasmid. It does not include the ...the PBAD promoter. If you already have a pBAD33-based plasmid expressing your gene of interest, all you ... created by the team (Fig. 2). These images are based on the concept of bacteriographs created about a...
  36. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    Published
    March 1, 2016, 3:30 p.m.
    ...which was technically challenging. Other homology based technologies We've described Sequence and Ligation...to SLIC and Gibson, there are yet more homology based assembly methods that have been described--CPEC ...store-bought cloning kits available which are all based on long-overlapping regions, have no requirements... these days. Gibson and the other long-homology based cloning methods are useful alternatives to the standard...assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009; 6, 343-345. PubMed PMID: 19363495...
  37. CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems

    Type
    Blog Post
    Published
    July 23, 2020, 1:20 p.m.
    ...off-target edits of base editors, which convert a nucleotide base pair to another base pair at a specific...Bhoobalan-Chitty Y, Peng X (2018) Anti-CRISPR-Based and CRISPR-Based Genome Editing of Sulfolobus islandicus...CRISPR activity is an important step towards CRISPR-based therapies and for more precise editing in the lab...
  38. CRISPR Cheat Sheet

    Type
    Blog Post
    Published
    Jan. 27, 2021, 2 p.m.
    ...DNA sequence. Base Editors: Modified versions of CRISPR nucleases that make single base changes in the...includes the details on advances in CRISPR including: Base editing Prime editing Anti-CRISPRs … you name it...
  39. Build Your CRISPR Vocabulary

    Type
    Blog Post
    Published
    July 2, 2024, 1:15 p.m.
    ... proteins (Cas9 shown here). The repeat sequence base pairs with the tracrRNA, and the spacer sequence...target DNA. tracrRNA: A “trans-activating CRISPR RNA” base pairs to the repeat section of the crRNA, forming...interchangeably with the term sgRNA. Seed sequence: The 8-10 bases on the 3’ end of an RNA guide (either crRNA or sgRNA...Sheet  Resources on the Addgene blog A Needle in a Base-Stack: Cas9 Structural Biology...
  40. Tag Your Favorite Yeast Genes with Ease

    Type
    Blog Post
    Published
    Nov. 19, 2013, 2:37 p.m.
    ...the modifying sequence flanked by approximately 50 base pairs of sequence homologous to the chromosomal ... expression in yeast. These tagging vectors are based on previously described pFA6a-link vectors and include...stability, and disruption of the tagged protein. Based on their findings, the authors recommend optimal...the artifacts sometimes associated with plasmid-based overexpression. Tim Formosa, at the University of...
  41. A Practical Approach to Choosing the B(right)est Fluorescent Protein

    Type
    Blog Post
    Published
    June 1, 2017, 2:30 p.m.
    ... Often, the choice for a fluorescent protein is based on the properties found in the literature or tabulated...is more sensible to compare fluorescent proteins based on their practical brightness in your experimental...mammalian cells this would be possible with CRISPR/Cas-based genome editing. Selecting a bright fluorescent protein...Amsterdam) where she develops genetically encoded FRET based biosensors.     Additional Resources on the Addgene...system as well as the imaging method, we recommend basing your selection on a small-scale head-to-head comparative...
  42. It’s Bioelectric! An Exciting Interdisciplinary Field of Research

    Type
    Blog Post
    Published
    Oct. 29, 2019, 2:52 p.m.
    ...the first thing that comes to mind. However, ion-based communication is not just for neurons. Non-neuronal...this too. It's just that for non-neural cells, ion-based communication happens at a slightly slower scale...cases even override genetically encoded patterning. Based upon trends in studies over the past several years...Chernet et al., 2016). There are many other light-based genetic tools for studying bioelectricity in vivo...desensitization in continuous light. ArcLight Q239 - A GEVI based on the fusion of the voltage sensing domain of a...
  43. Viral Vectors 101: Biosensors

    Type
    Blog Post
    Published
    July 6, 2023, 1 p.m.
    ... in the presence of calcium. This includes FRET-based sensors such as Twitch. While calcium is king in...Neurotransmitters Many canonical fluorescent as well as FRET-based biosensors have been engineered to detect a wide...ATP ratio. Most of these sensors are fluorescence-based and have been optimized for a wide-range of uses... pair on actinin will produce different signals based on changes to the structure of actinin in low vs...Biosensors Illuminating Epigenetics with a FRET Based Biosensor Fluorescent Proteins 101: Fluorescent...
  44. Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity

    Type
    Blog Post
    Published
    Oct. 11, 2018, 12:30 p.m.
    ... sites in an orientation that results in 5’ four base overhangs after cutting. The destination vector ...IIS restriction enzyme cutting results in 5’ four base overhangs complementary to the overhangs of the ...Single insert cloning efficiencies were measured based on the acquisition of antibiotic selectable markers...markers. The 5-, 12- and 24-assembly systems were based on the ability to correctly assemble a lacI/lacZ... over 50 years, prefers ligation of Watson-Crick base pair substrates, it demonstrates significant activity...cohesive end ligation by this enzyme for all 3- and 4-base overhang sequences under standard reaction conditions...
  45. AAVs in Retinal Gene Therapy

    Type
    Blog Post
    Published
    July 31, 2018, 12:55 p.m.
    ...the field of gene therapy, more specifically AAV-based retinal gene therapy. The year was 2001 and I started...with the death of Jesse Gelsinger in an adenovirus-based gene therapy clinical trial gone horribly wrong....cell) therapies. One may wonder why the first AAV-based gene therapy treatment for a genetic disease approved...forward with ocular gene therapy The pipeline of AAV-based ocular gene therapies is impressive: clinical trials...for prevalent diseases - to name a few. CRISPR-based tools are also particularly well suited to be combined...and Wu Z., 2018. In vivo Applications of CRISPR-Based Genome Editing in the Retina. Front Cell Dev Biol...
  46. Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR

    Type
    Blog Post
    Published
    July 2, 2020, 12:40 p.m.
    ...transcription. Sometimes enhancers are located thousands of base pairs away from the gene they regulate, but are ...detected with chromosome conformation capture (3C) based methods, such as 5C or Hi-C (Han et al., 2018). ...increase the resolution, the Xu lab created a dCas9-based CAPTURE (CRISPR Affinity Purification in situ of...DNA that are far away from each other in terms of base pairs, but close together in terms of where they...dCas9 and gRNA expression. Additionally, CAPTURE 3C-based sequencing requires a large number of cells (~5x107...Han, J., Zhang, Z., & Wang, K. (2018). 3C and 3C-based techniques: the powerful tools for spatial genome...
  47. Plasmids 101: Control Plasmids

    Type
    Blog Post
    Published
    April 9, 2015, 3:29 p.m.
    ...any plasmid-based experiment, untreated cells should be included as these provide the baseline/standard ...one thing should be present in every experiment-based investigation of a hypothesis: proper controls! ...the various controls that can be used for plasmid-based experiments. What are control plasmids? Generally...the abstract and follow an example of a plasmid-based experiment, delineate its requisite control plasmids...interpretation of the results. Typically, plasmid-based experiments employ transfection, negative, positive...completely. These controls may or may not be plasmid-based depending on the experimental needs. Using our experiment...
  48. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

    Type
    Blog Post
    Published
    April 9, 2019, 1:13 p.m.
    ...homodimerization of FPs targeted to the endoplasmid reticulum based on the appearance of OSER whorls. This assay is ...random insertions can be generated with a transposon-based strategy (Sheridan, 2002). Avoiding protein over-expression...first express the fusion protein from a plasmid-based system to assess whether the chimera is still functional...verified and it can be compared to what is expected based on immunofluorescence or properties of the protein...independent of GDI-mediated extraction and actin-based trafficking." PLoS biology 13.4 (2015): e1002097...Klarenbeek, Jeffrey, et al. "Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness...PMID: 30886412. Mastop, Marieke, et al. "A FRET-based biosensor for measuring Gα13 activation in single...
  49. A Control for All Seasons

    Type
    Blog Post
    Published
    Oct. 5, 2023, 1:15 p.m.
    ...controls for antibody-based experiments Primary antibody specificity Antibody-based assays have their own...remains the same.    Figure 2: Microscopy-based antibody experiments should include a primary antibody....   Application-specific controls Some antibody-based applications have very specific controls, which ...you to detect dead or dying cells so that you can base your analysis on living cells only (for example,...some of the more critical controls for antibody-based experiments, it is by no means an exhaustive list... more information about antibodies and antibody-based experiments, check out these blog posts.       ...protocols elicited the expected response and have a baseline with which to compare the experimental group. ...
  50. Comparing Cas9 to NgAgo: Can the Argonautes Best CRISPR?

    Type
    Blog Post
    Published
    June 9, 2016, 2:30 p.m.
    ...guides employed by Cas9 or Cpf1. These short, 24 base DNA guides may be more user-friendly, since they...working with small capacity viral vectors such as AAV. Base removal RNA-guided CRISPR enzymes cause double-stranded...unfavorable. In a natural context, it’s hypothesized that base removal helps prevent the invading genome from “...property could be an advantage. However, repair after base excision would alter the target sequence and preclude...
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