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We narrowed to 810 results for: nin

Showing: 51 - 100 of 810 results

  1. Fluorescent Protein Travel Awards - Protein Variants, a Serotonin Sensor, and an Artificial Leaf Replica System

    Type
    Blog Post
    Published
    June 4, 2019, 1:02 p.m.
    ...encoded fluorescent sensor for serotonin. According to Unger, “serotonin is a well-known but poorly studied...medium-throughput screening, she increased the affinity of the sensor for serotonin by over four orders...leads to the release of serotonin (measured using the newly developed green serotonin sensor). In preliminary...use the serotonin sensor in sleep-related studies and in assays for measuring human serotonin transporter...Chiasson explains. Elizabeth Unger: Developing a serotonin sensor The goal of Elizabeth Unger’s postdoctoral...there are no known bacterial proteins that bind serotonin. Unger redesigned the binding pocket of the periplasmic...periplasmic binding protein so that it would recognize serotonin. Using a combination of computer modeling, directed...

  2. Hot Plasmids - March 2019 - Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable Proteins

    Type
    Blog Post
    Published
    March 19, 2019, 1:31 p.m.
    ...2in1 vector cloning system Article contributed by Michelle Cronin  Listen to the 2in1 cloning podcast segment...traditional Gateway™ cloning, the GOI first needs to be cloned into an entry vector. The 2in1 cloning system contains...restriction digest cloning to introduce the GOIs into the 2in1 vector instead of the GatewayTM cloning BP reaction...Recently, the Grefen lab introduced the “2in1” vector cloning system - a Gateway compatible approach to simultaneously...the GOI occurs through recombination. The 2in1 cloning system is well suited for protein-protein interaction...coexpression. By introducing a single plasmid containing both GOIs, expression levels are more evenly ...controlled. The Grefen Lab has utilized the 2in1 cloning system to generate a series of plasmids to study...

  3. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

    Type
    Blog Post
    Published
    June 2, 2020, 1:15 p.m.
    ... Figure 1: Initial steps in the CRISPR-pooled screening using sgRNAiBAR. Image from Zhu et al., 2019.  ... Peking University has developed a new CRISPR screening method that makes use of guide RNAs with internal... After infecting cells with sgRNAiBAR library containing lentiviral vectors the iBARs can be detected ... annotated human gene, it enables genome-wide screening with a high MOI, which is beneficial for a low... calcineurin dynamics and can serve as a drug-screening platform to identify new antifungals that target... assay, and are not suited to high throughput screening, calcineurin dynamics measurements, or monitoring... that this method is compatible with dead cell staining using propidium iodine (PI), allowing for calcineurin...

  4. Plasmids 101: Common Lab E. coli Strains

    Type
    Blog Post
    Published
    Nov. 7, 2014, 2:56 p.m.
    ...purpose: fast growth, high-throughput cloning, routine cloning, cloning unstable DNA, preparing unmethylated...Streptomycin MC1061 derivative. General cloning and storage, blue/white screening, leucine auxotroph. F- endA1 recA1... DH5alpha   General cloning and storage of common plasmids, blue/white screening. F- endA1 glnV44 thi...glnV44 λ- JM109   General cloning and plasmid maintenance, blue/white screening, partly restriction-deficient...NEB Stable   For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences...JM109-derived. For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences...from HB101. For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences...

  5. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    Published
    July 20, 2021, 1:15 p.m.
    ...Direct staining is quicker than indirect staining and eliminates potential non-specific staining that may...can use both direct and indirect staining methods. In direct staining, the primary antibody against a ...intracellular staining where binding of large immune complexes may be hindered. Indirect staining can be beneficial...also keep their targets in mind when planning fixation. If staining both cell surface and intracellular...control is critical for determining the background level of non-specific staining.  If you’re using antibodies...-specific staining associated with the primary antibody. When following an indirect staining protocol,...omitted from the staining protocol. This control allows you to detect background staining arising from the...

  6. How to Be an Excellent Trainee

    Type
    Blog Post
    Published
    Sept. 26, 2023, 1:15 p.m.
    ...students, rotation students, joining a new lab at any career stage, learning a new technique while visiting...participant in the training. In this blog we will review the preparation and planning you can do to build...true for training in the lab! If you don’t do proper preparation and pay attention during training, then ...questions to your training! All of these things will help you get more out of the in-person training component...need to double-check a detail! Training follow up After your training session (ideally in the next few... the information in your training. Bench mentor-mentee relationships Training can be a very fulfilling...visiting a lab, or maybe even training on a new technique with a coworker. Being a new trainee (even if you...

  7. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    Published
    Jan. 6, 2015, 5:21 p.m.
    ...constructs? A new cloning system combining the advantages of Golden Gate and Multisite Gateway cloning methods ...that you need. The Golden GATEway cloning kit simplifies the cloning process for complex DNA constructs...vectors via traditional restriction enzyme cloning, TA cloning, or annealing of oligonucleotides. These ...efficient and flexible cloning process provides improvements to classical cloning methods, particularly... MoClo modular cloning system Synthetic biologists have developed a modular cloning strategy, MoClo,...assembly steps. Before beginning, scientists can insert fragments of DNA containing basic parts (promoters... of interest are retained after SLIC cloning. The general cloning strategy described in the associated...

  8. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    Published
    Dec. 23, 2015, 3:30 p.m.
    ...building block for further chaining. The MXS-chaining kit contains 5 empty chaining vectors plus a destination... 19; 22(2):169-74. High efficiency cloning for enzyme screening Dr. David Ackerley’s lab at the University...expression containing a variety of N- and C- terminal tags. The Santalucía Lab has developed a cloning toolkit...Multisite Gateway cloning. A desired insert is cloned using traditional restriction enzyme cloning to create ...(5):493-8. MXS cloning platform for imaging and flow cytometry applications Designing customized fluorescent...Neveu laboratory designed a new iterative, chaining-based cloning method that simplifies the process of constructing...with MXS-chaining. Analogous to modular assembly methods, such as BioBricks, the MXS-chaining method uses...

  9. Pushing the Limits of DNA Assembly

    Type
    Blog Post
    Published
    Sept. 6, 2017, 1:01 p.m.
    ...any well-executed cloning endeavor, NEBuilder requires upfront planning. This cloning methodology requires...? In the context of cloning, DNA assembly refers to a method of physically joining multiple fragments ... the joining reaction. BioBrick Assembly will leave scar regions at the site of fragment joining, but ... for seamless joining of multiple mutations spread over thousands of base pairs. Cloning sgRNAs Generating... More about Gateway Cloning Gibson Assembly and Other Long-Homology Based Cloning Methods Screen Plasmids...end of a fragment. This means that fragments (containing regions of homology) cut from plasmids via restriction...the regions of homology. A powerful resource for planning an NEBuilder HiFi DNA Assembly mix reaction is...

  10. 27 Hot Plasmids from 2016

    Type
    Blog Post
    Published
    Dec. 22, 2016, 3:03 p.m.
    ...process of cloning, combining, and testing these different parts can be quite arduous. The cloning process...of a good screening experiment can be found on our blog post entitled “Genome-wide Screening Using CRISPR...Moduclar Cloning (CIDAR MoClo) Parts Kit. This Kit consists of a variety of plasmids containing many different...compatible with both standard Gateway cloning and the FNLCR Combinatorial Cloning Platform (CCP); the latter of...terminators for easy cloning of genes and promoters. In addition to USER cloning, this toolkit is also...GPCRs in the human genome are orphan receptors meaning their ligands are unknown. Identifying new ligands...approach termed Parallel Receptor-ome Expression and Screening via Transcriptional Output-TANGO (PRESTO-TANGO...

  11. Plasmids 101: Plasmid Incompatibility

    Type
    Blog Post
    Published
    March 31, 2020, 1:15 p.m.
    ....  The partitioning problem Plasmid incompatibility also occurs because of plasmid partitioning (Schumacher...within one cell have the same replicon and/or partitioning system. Let’s start with the replicon- the part...to initiation replication. However, in iteron-containing plasmids,  RepA can also bind to its own promoter...a concern with low copy plasmids, though the partitioning problem does occur in high copy plasmids (Diaz...are several different versions of the plasmid partitioning system in the bacteria, but generally it consists...For high copy plasmids, incompatibility due to partitioning is similar to incompatibility due to having ...loss (Diaz et al., 2015).  Figure 2: The partitioning problem. For low copy plasmids, the main ...

  12. Plasmids 101: Colony PCR

    Type
    Blog Post
    Published
    May 12, 2016, 2:30 p.m.
    ...Molecular cloning requires some method of screening colonies for the presence of an insert. Traditionally...then you might consider designing orientation-specific primers. Blunt end cloning is an example of when ... Addgene Blog Learn to do Restriction Cloning Try SLIC cloning Learn about Gibson Assembly Additional...things to consider when performing colony PCR.   Designing colony PCR primers The first and perhaps most ...most important step to colony PCR is designing primers. There are 3 strategies for primer design: 1) insert-specific...backbone. This type of primer pair is also great for screening clones created with the same backbone but that... When you design primers to anneal outside the cloning site, it doesn’t matter what the sequence of the...

  13. Which Fluorescence Microscopy Techniques is Best for Me?

    Type
    Blog Post
    Published
    Oct. 10, 2017, 1:57 p.m.
    ...for an example image). Point Scanning Confocal Microscopy Point Scanning Confocal Microscopy was the first...microscopy technique to incorporate optical sectioning. Optical sectioning refers to the ability to extract light... Confocal Microscopy (Spinning Disk) Parallelized Confocal Microscopy (Spinning Disk) increases the speed...to enhance performance. In spinning disk microscopy, a metal disk containing a number of holes rotates ...samples always require some form of optical sectioning. Point scanning confocal microscopes (see above) often...volumes to enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy...excitation light to a point in the sample and raster scanning the point to build up a final image pixel by pixel...

  14. CRISPR 101: Any Base Transversion Editors

    Type
    Blog Post
    Published
    April 1, 2025, 1:15 p.m.
    ...transitions are edits between the purine bases (adenine, guanine, and hypoxanthine) or between the pyrimidine...glycosylase (MPG), also known as alkyladenine DNA glycosylase (AAG). When adenine base editors (ABEs) were originally...BioRender.com. Inosine excision leads to the first adenine transversion editors Uracil excision is the most...endogenous enzyme is not efficient enough to hinder adenine base editing. Once base transversion editors were... able to edit A → Y (Y = T or C) in 67.17% of screening cells (Tong, Wang, et al., 2023). Based on this... team used rational mutagenesis to produce the adenine transversion editor AYBEv3. This tool still retained...A → C product purity of 48%. Like CGBEs, these adenine transversion editors produce a lot of indels (up...

  15. Is this the right place for me? 8 tactics for choosing a lab

    Type
    Blog Post
    Published
    Oct. 2, 2018, 12:56 p.m.
    ... analytic scientists seem to toss data-driven reasoning out the window when making decisions. We think...decisions without much logic - even choosing a training lab where we will spend the next 6 years. Know...very communal. That didn’t suit my collaborative learning style at all. Finally I rotated in a small lab...about this lab’s reputation? If you are choosing a training lab, I strongly suggest you complete a work rotation... the same field taking projects with them and remaining close colleagues of the PI? Or do lab alumni leave...they “needed it more”. My friend didn’t heed this warning and profoundly regretted it when the same thing...year later. I hear too many science trainees get warnings and still think “but that won’t happen to me, ...

  16. A Conference By Postdocs For Postdocs: Future of Research

    Type
    Blog Post
    Published
    Sept. 2, 2014, 3:05 p.m.
    ...of scientist training in the US and around the world. There are rumors that we are training too many scientists...opportunities after training. One of the best recent discussions of this apparently worsening crisis recently...the way we view the graduate school and postdoc training years. There is no doubt that there are too many...academia. Certainly funding for academic research and training is getting tighter and competition is fierce. ...sufficiently prepared during their 6-12+ (!) years of training to get jobs. They are not exposed to the vast ...radical (and some not so radical) suggestions for defining the problem and for finding solutions. Even better...meeting new people by working together on the event planning and execution. I am seeing many similar examples...

  17. RANbodies: Reporter Nanobody Fusions

    Type
    Blog Post
    Published
    April 10, 2018, 12:56 p.m.
    ...reporter. The only caveat is that HA staining is weaker than reporter staining. Picking the right reporter for... them. HRP RANbody staining results in a ≥10-fold increase in signal over staining with a primary/ labeled... sensitive than HRP-RANbody staining Requires secondary antibody staining HA Tagged GFP, RFP, H2A2B ... IgY antibody Staining cultured cells and tissue sections Great for multicolor staining of tissue since... sensitive than HRP-RANbody staining Requires secondary antibody staining GFP, RFP, H2A2B   Are you...smaller size of RANbodies compared to antibodies. Staining with multiple HRP-RANbodies is indistinguishable... domains are especially useful for multi-color staining of tissue because they have limited cross-reactivity...

  18. New Optogenetic Tools for Cytoskeleton and Membrane Control

    Type
    Blog Post
    Published
    Sept. 28, 2023, 1:15 p.m.
    ...Figure 1: Opto-katanin induces fast, localized microtubule severing. A) Schematic of Opto-katanin constructs...microtubule-severing protein, Katanin is of course named for the samurai sword called a katana. Opto-katanin consists of...use. And now, onto our optogenetic tools!  Opto-katanin: a light-powered microtubule-cutting sword Microtubules...tool for localized microtubule disassembly, Opto-katanin (Meiring et al., 2022), that overcomes many of ...EB3N-VVDfast-iLID (anchor) and SspB-mCherry-p60 (katanin) are recruited to microtubules under blue light...from the microtubule body. B) Schematic of Opto-katanin’s use in cells. Images adapted from Meiring et al...microtubules and iLID-SspB to control the recruitment of katanin to that anchor. So, you’ll need to express both...

  19. Technical Design of a Western Blot

    Type
    Blog Post
    Published
    Aug. 22, 2024, 1:15 p.m.
    ... thought about running one, you’ll know there’s a lot of choices to make when designing the experiment...for SDS-PAGE running conditions and will cover most western blot use cases. If you’re running native (non-reduced...of gels  Gel Type Protein sizes Running buffer Running conditions Pros Cons Tris-glycine...Figure 1: The technical decisions to make when designing a western blot.  Psst! Looking for a western...lyse and denature them. If you are intentionally running non-denatured proteins, in what is known as a native...proteins. For this post, we are assuming that you are running a denaturing blot.  There are a number of lysing...cytoplasm or whole-cell lysate. A harsher, SDS-containing lysis buffer, like RIPA, should be considered...

  20. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    Published
    June 28, 2016, 10:59 a.m.
    ...genomic fragment containing the sgRNA target sequence can be placed in multi-cloning site (MCS) between...floating around, and many pre-designed plasmids containing these variants ready for purchase. But what is...post will help get your in vivo research up and running as soon as possible! Selecting your target CRISPR...has been quite successfully adapted for use in screening experiments as well). Therefore, if you’ve started...to attack this gene. Should you cut it at the beginning of the coding sequence to delete the entire gene...previously validated gRNAs at Addgene. In vitro screening for the best gRNA Figure 1: Validation ...plasmid was co-transfected with pX330 plasmids containing sgRNA sequences into HEK293T cells. The fluorescence...

  21. When Fidelity Matters: A frank discussion about ligase fidelity

    Type
    Blog Post
    Published
    March 6, 2018, 1:49 p.m.
    ...including vector-insert joining for recombinant plasmid construction (restriction cloning), adaptor ligation...Learn New Plasmid Cloning Techniques Pushing the Limits of DNA Assembly Plasmid Cloning by PCR Resources...ligases are enzymes that seal breaks in DNA by joining 5 ́-phosphorylated DNA termini to 3 ́-OH DNA termini... is highly active in nick ligation and DNA end-joining, but will also efficiently ligate many undesirable...undesirable structures, including substrates containing gaps or DNA base pair mismatches (8-11). For some applications...applications, there cannot be any end-joining activity, and for others, there is a need for the exclusive...DNA Ligase, which only reacts with substrates containing no gaps, and will not join any fragments end-...

  22. Antibodies 101: Choosing the Right Isotype

    Type
    Blog Post
    Published
    April 6, 2023, 3 p.m.
    ...secondary antibody) approach?  Staining for multiple targets When staining multiple targets indirectly, ...for a four-antibody panel, staining in parallel    In short, when staining multiple targets, look for...will recognize and bind to your primary antibody. Manning et al. tested a variety of commercially available...there was a detection bias of IgG2a>IgG2b>IgG1 (Manning 2012).      Figure 1: Broadly reactive...depends on the application and target. If you are running a basic western blot and probing a single target...mouse tissues, causes a high degree of background staining. This occurs because the anti-mouse secondary ...the tissue sample. There are species-on-species staining protocols that use antibody fragments to block...

  23. Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity

    Type
    Blog Post
    Published
    Oct. 11, 2018, 12:30 p.m.
    ...More complex 24-fragment assembly Single insert cloning efficiencies were measured based on the acquisition...Antibiotic selection allowed for high throughput cloning efficiency measurements. We performed additional...suppression of background by antibiotic selection. Screening of transformants by colony PCR confirmed the insertion...presence of every module, destination construct and functioning Type IIS restriction enzyme and DNA ligase, any...obtained if any single component was omitted. Screening for successful Golden Gate Assembly All Golden...researchers can plate larger volumes of the outgrowth containing the transformants onto the selection plate. Figure... Ligase, the mainstay of most biotechnological cloning efforts for over 50 years, prefers ligation of ...

  24. Transferable Skills Guide: External Collaborations

    Type
    Blog Post
    Published
    April 19, 2018, 1:25 p.m.
    ...that many researchers feel that they lack formal training in a variety of transferable skills. At Addgene...huge asset! As is true for any skill, there is a learning curve for collaborating and it is always a good...perfect platform for practicing initiative and for training in teamwork and collaboration management. For ... email and social media (see the section on “Maintaining Relationships” below). Expand: Collaboration ...complex projects. It is also fun! I really enjoy learning from others and meeting people with different ...was very difficult and somewhat awkward in the beginning. But, as so often happens, one gets better with...people outside the university within the context of learning and, in my experience, mistakes and insecurities...

  25. Antibody Validation for Flow Cytometry

    Type
    Blog Post
    Published
    Sept. 10, 2024, 1:15 p.m.
    ...populations, as well as a pragmatic approach to determining the selectivity of antibodies for other sample...found that some antibodies perform well in a PFA-saponin fixation and permeabilization protocol, but very...usually test 3 different fix/perms (methanol, PFA-saponin, PFA-triton). The fix/perm solutions can make a... your antibody. When compared, 4% PFA and 0.1% saponin; 4% PFA and 0.1% Triton X-100; and methanol, we... and fixed in 4% PFA and permeabilized in 0.1% saponin. 400,000 cells were stained with the indicated ...CoraLite® Plus 647 secondary antibodies. Antibody staining was quantified using the Attune NxT Flow Cytometer...Cytometer with representative images showing the staining intensity in the KO population (pink histogram)...

  26. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    Published
    Sept. 17, 2024, 1:15 p.m.
    ...causing it, try making fresh running buffer. Old or improperly made running buffers can cause all kinds...transfer worked with Ponceau S staining or other reversible protein staining immediately after your transfer...troubleshoot a western, you’ll need to be familiar with running a gradient, which will allow you to optimize the...can help answer your particular question before running one.  Good on gradients? Excellent! Now, let’s ...subcellular compartment, you may want to try an SDS-containing detergent like RIPA. You can try a few different...V/cm of gel), your gel may be overheating. Try running it in a cold room or putting ice packs in and/or...kinds of odd issues. If your gel is running properly but you’re not seeing sufficient resolution in the bands...

  27. Making CRISPR Plasmids Using Fragmid

    Type
    Blog Post
    Published
    Aug. 29, 2024, 1:15 p.m.
    ... them not working, or spend a few weeks designing and cloning the vectors you actually want. But now you...Plasmids 101: Modular Cloning Addgene's CRISPR Guide Plasmids 101: Golden Gate Cloning...designed for a quick, plug-and-play approach to cloning CRISPR vectors (McGee et al., 2024). It reduces...truly is plug-and-play. Because of its modular cloning approach, it’s easy to switch out reporters, Cas... overview and timeline of the Golden Gate (GG) cloning approach. Individual modules and a sample ligation...them into a destination vector via Golden Gate cloning, perform an exonuclease cleanup, transform, plate...experiment. As always, we recommend checking your cloning via a miniprep and restriction digest, and then...

  28. CRISPR 101: Multiplex Expression of gRNAs

    Type
    Blog Post
    Published
    Sept. 10, 2020, 11:45 a.m.
    ...PCR amplified for cloning into one of three Zeamays codon-optimized Cas9-containing binary vectors. The...introduce you to some of your potential options and cloning methods, but please scroll down for plasmids suitable... can be inserted using Gibson Assembly or SLIC cloning methods. A BsaI-based E. coli multiplexing plasmid...together and drive ordered assembly into a Cas9-containing destination vector. Note: depending on which ...four unique kanamycin-resistant plasmids, each containing a different gRNA target sequence downstream of...digested using BsmBI and ligated into Cas9 or dCas9-containing destination vectors. Destination vector options...), and dCas9-VP64 (transcriptional activator)-containing plasmids. Each destination vector contains GFP...

  29. Plasmids 101: Secondary Nanobody Toolbox

    Type
    Blog Post
    Published
    Feb. 27, 2018, 2:04 p.m.
    ...comparison of this one-step staining with nanobodies vs two-step staining with antibodies. Nanobodies...before application to the sample. Staining with this multi-color staining workflow yields similar localization... fusion with a reporter enzyme. For multicolor staining, careful consideration must be used when selecting...different IgG subclasses of 1° for multi-color staining Can be expensive Nanobodies   Recombinant...together. At the bench, this results in a shorter staining protocol because the primary antibody can be “...Nanobodies also simplify multicolor staining experiments because they don’t require using primary antibodies from...below, triple colocalization is possible even when staining cells with three IgG1 antibodies. Antibodies were...

  30. 9 tips for a successful postdoctoral experience

    Type
    Blog Post
    Published
    Oct. 9, 2018, 12:21 p.m.
    ...number of challenges ranging from long periods of training to limited job opportunities in academia - the...the main reason most people enter postdoctoral training. Similarly, there are several factors to consider...about how to get the most out of your postdoctoral training experience. If you're going to commit to doing...doing a postdoctoral fellowship, what are you signing up for and how can you do it well? Below, I briefly...and create a timeline for achieving these goals. Planning is often more easily done in reverse. Assuming...before the position would begin- for you, at the beginning of Year 5. Now work backwards. To publish a submitted... critical to get an experimental system up and running very early on. Once your system is working, you...

  31. Plasmids 101: Gram Negative and Positive Bacteria

    Type
    Blog Post
    Published
    Aug. 2, 2022, 1:15 p.m.
    ...gram negative bacteria, staining them red or pink. At the end of the staining procedure, gram positive...Streptococcus pneumoniae, while the alcohol used in the staining process bleached another, Klebsiella pneumoniae...expanded on Gram’s discovery, adding a second stain, safranin, as a counterstain to help visualize the bleached...and immediately rinse. Flood the smear with the safranin counterstain for 30 - 60 seconds. Rinse the slide...positive (purple) and Gram negative (red/pink) staining. Image credit: Mark Perkins https://www.flickr.com...check out these resources.   Bruckner, M. Gram Staining. https://serc.carleton.edu/microbelife/research_methods...creating a complex within the peptidoglycan layer, staining the cell purple. When the alcohol is added, it...

  32. CRISPR 101: Validating Your Genome Edit

    Type
    Blog Post
    Published
    Nov. 3, 2022, 12:15 p.m.
    ...are ready to begin the screening process. Want to set yourself up for a screening success? Before you start...’s best to do some planning in the design phase of your experiment for a screening strategy. Restriction...looking for your edit of interest. For all of the screening strategies outlined below, the technique can be... isn’t a multiple of three will do. A popular screening method for knockouts is Tracking of Indels by ...frequency of your sgRNA-Cas9 combo. Pro tip: When designing your PCR, ensure there is at least ~200 base pairs...deletion large enough that it can be visualized by running your PCR on a gel. It’s still paramount that you...using a dual guide system.   Fig. 2: Size screening for large deletion events.   Validating knock-in...

  33. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    Published
    Aug. 2, 2016, 2:30 p.m.
    ...functional analysis of proteins (to perform alanine scanning mutagenesis or targeted substitution of key... the plasmid containing the mutation). Similar results can be obtained with PCR screening using the primers...manner. The approach can be used in conventional cloning (to introduce or remove restriction sites), in ...final circular plasmid product pictured here. B) Screening of recovered plasmids for the mutation of interest... vastly facilitates the subsequent process of screening for succesfully mutated clones. Forward and reverse...Taq generate A-overhangs (a feature used in TA-cloning). Such non-complementary bases at the 3’-end interfere...can also form colonies under these conditions. Screening If a restriction site was introduced (or ablated...

  34. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    Published
    Sept. 29, 2016, 2:30 p.m.
    ...of whole phages as therapeutics, which is also gaining a lot of new traction lately (1, 2), depends on...varied by the cell and whose structures are just beginning to be understood. Therefore, in addition to their...identifying and characterizing sugars and sugar-containing molecules in molecular biology labs (for a review...binding proteins are hard to find Sequencing and cloning phage DNA can be difficult, in part because phages...Nebulization involves forcing DNA through a small opening using compressed nitrogen gas; this shears DNA ...RBP gene without causing issues associated with cloning large fragments. After fragmentation, repair sheared... colonies using primers bordering the multiple cloning site of the vector. In order to reduce the chances...

  35. PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs

    Type
    Blog Post
    Published
    June 25, 2024, 1:15 p.m.
    ...especially with my deep learning-wizard colleague Ahmed Allam. Continued training led to the recent development...High-throughput screening of pegRNA efficiency and building prediction tools with machine learning (PRIDICT,... scaffold sequence, and an extension sequence containing a primer binding site and a reverse transcription...al., 2024). The additional datasets used in the training of PRIDICT2.0 make this model more suited for ...your target. Figure 3: High-throughput screening of the effect of chromatin on prime editing efficiency...prediction tool (ePRIDICT). In summary, machine learning-based prime editing prediction models (such as...biology. His expertise includes applying machine learning techniques to make sense of biology, with a special...

  36. Course-Based Undergraduate Research Experiences (CUREs)

    Type
    Blog Post
    Published
    Feb. 9, 2023, 2:15 p.m.
    ... an education . . . is not the learning of many facts but the training of the mind to think something ...take a different approach. His own most impactful learning experiences happened in the lab, and he wanted... work and become more actively involved in the learning process. As trainees become more comfortable with...interest in scientific career paths while also broadening access to experiences that promote professional...trading structure and predictability for improved learning outcomes and the opportunity to make new discoveries...teaching style, with the former offering excellent training in improvisation and the latter making him a more...by measuring NADH production.   CUREs are beginning to transform undergraduate education in the sciences...

  37. Typing CRISPR Systems

    Type
    Blog Post
    Published
    March 18, 2025, 1:15 p.m.
    .... P., White, M. F., Yakunin, A. F., Garrett, R. A., Van Der Oost, J., . . . Koonin, E. V. (2015b). An ... M. P., Venclovas, Č., White, M. F., Yakunin, A. F., . . . Koonin, E. V. (2019). Evolutionary classification...classification system for the array of CRISPR technologies. Learning the general principles of this classification ...S., Oshiro, R., Makarova, K. S., Macrae, R. K., Koonin, E. V., & Zhang, F. (2023). Uncovering the functional...Chylinski, K., Makarova, K. S., Charpentier, E., & Koonin, E. V. (2014). Classification and evolution of ...1314. https://doi.org/10.1134/s0006297921100114 Koonin, E. V., & Makarova, K. S. (2019). Origins and evolution...20180087. https://doi.org/10.1098/rstb.2018.0087 Koonin, E. V., Makarova, K. S., & Zhang, F. (2017). Diversity...

  38. Editor's Choice, September 2016

    Type
    Blog Post
    Published
    Oct. 7, 2016, 4 p.m.
    ...for Host Binding Proteins & Running for Rare Disease, Running for FOP, Running for AJ I have two picks for...the post. The second post, “Running For Rare Disease, Running for FOP, Running for AJ,” was contributed ... we’re raising money for a cause while running. I love running and this event gives me the opportunity...sitting in the San Francisco International Airport listening to the Lion King soundtrack and writing this post...Project That’s it for now. As I indicated in the opening paragraph, I’m currently on my way back from a ...

  39. Fluorescent Tagging of Endogenous Genes with SapTrap

    Type
    Blog Post
    Published
    Oct. 12, 2017, 2:26 p.m.
    ...generate gRNAs and homology arm repair templates containing the tag for each insertion. Also, it remains ...simultaneously introduce a selection marker for screening modified strains. With SapTrap, the user first... (Fig. 1, Step 1). There is no need for PCR or cloning, as digestion of the destination vector with SapI...SapTrap includes a prebuilt donor plasmid library containing several types of fluorescent (EGFP, tagRFP, mCherry...selectable marker (floxed Cbr-unc-119) for easy screening of the insertion event, and a variety of connector...hundreds of genes, generating powerful genetic screening libraries. Additional protein tagging systems ...reading frame of the insertion, and 3) a vector containing the desired tag, which can be obtained as a universal...

  40. PITChing MMEJ as an Alternative Route for Gene Editing

    Type
    Blog Post
    Published
    Feb. 23, 2016, 3:30 p.m.
    ...microhomology-mediated end-joining)? MMEJ, a form of alternative end-joining, requires only very small ...research, you know all about using non-homologous end-joining (NHEJ) to make deletions or homology-directed repair...month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction There...repair template is present. HDR also presents a cloning dilemma - the longer the homology, the more efficient...with longer homology arms comes more time spent cloning. In contrast, the short homology required by MMEJ... regions of homology, which facilitates easier cloning. An overview of PITCh. The PITCh plasmid...inserted into the PITCh vector via In-Fusion or SLIC cloning. Step 2: Design an insertion locus-specific gRNA...

  41. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    Published
    Jan. 3, 2019, 1:35 p.m.
    ...defective allele Ym and you need to convert an adenine (A) to a guanine (G)? You may wonder if a CRISPR-base editor...molecular components. Adenine base editors for plant We developed plant adenine base editor (ABE) vectors...with the desired outcome are not pictured. Adenine base editors (ABE) What if you have another gene...deaminases' which would theoretically give rise to an adenine base editor. A massive protein engineering effort...an adenosine deaminase that can act on DNA for adenine base editing. They first created a defective chloramphenicol... (H193Y). The reversion of this mutation by an adenine base editor would restore antibiotic resistance...into E. coli harboring the defective CamR gene. Screening for surviving colonies and subsequent rounds of...

  42. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    Published
    July 13, 2023, 1:15 p.m.
    ...pooled libraries, from cloning a pooled library to viral production to screening. Many of Addgene’s pooled...libraries here at Addgene, including cDNA, barcoding, screening, and gRNA libraries. Most of the pooled libraries...determined, larger lentiviral preps can be used for screening-scale transduction of cells, resulting in cell...library. These cells can then be used for genetic screening, followed by sequencing and further data analysis...quality control our viral preps, we recommend determining the functional (infectious) titer once you've...as seeding density and reagent concentration.  Screening time Phew! You are now (finally!) ready to use...hesitate to email us at [email protected]. Happy screening! This blog was written by Addgenie Hannah Dotson...

  43. Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

    Type
    Blog Post
    Published
    Nov. 1, 2018, 12:35 p.m.
    ... in plasmid cloning Perhaps the most widely used application of CcdB is GatewayTM cloning, where the ccdB...toxin is stable while the antitoxin is unstable, meaning that the cells must continually produce antitoxin... not inherit plasmid due to improper plasmid partitioning, it will still inherit some toxin and antitoxin...counterselectable marker in the destination vector. Successful cloning occurs when the ccdB gene is replaced by the gene...modifications in the yeast Pichia pastoris. By combining both positive and counter selection in one plasmid...plasmid, they were able to create an efficient cloning tool. The plasmid pKSCTMF contains the mazF gene...retained in less than 10% of cells while plasmids containing the plasmid addiction system were retained in...

  44. RUBY-Red Siliques

    Type
    Blog Post
    Published
    March 1, 2022, 2:15 p.m.
    ...to select against CRISPR positive plants, but screening these can be challenging and requires specialized...requiring the destruction of plants or tissue for GUS staining or are not suited to studying large samples. RUBY...sites. These convert the amino acid tyrosine to betanin, a member of a class of pigments primarily found...order Caryophyllales, such as cacti and beets. Betanin belongs to a different chemical family than the...purple plant pigments found in all other plants, anthocyanins.   Figure 1: An adult plant expressing...selecting transformation events and am currently planning to use it to simplify my work with an Arabidopsis... to study these mutants.   Fig. 2: (A) Betacyanin is produced from tyrosine by the activity of ...

  45. Antibodies 101: Avoiding the Mouse on Mouse Mess in IHC

    Type
    Blog Post
    Published
    Sept. 21, 2023, 1:15 p.m.
    ...are crisp, bright, and lacking in non-specific staining can be a challenge in the best of cases. But it... antibody, resulting in significant background staining (Figure 1A). Not all tissues will have the same...researchers can get away with species on species staining with no problem! But to be sure that your secondary...experiments. If you do find high levels of background staining by the secondary, then the following three strategies...help!     Figure 1: Species on species staining can be a problem because the secondary antibody...that you can significantly decrease background staining by mixing your primary and secondary antibodies...are a few common solutions to avoid background staining when doing species on species IHC. (A) One solution...

  46. With an Eye Towards the Future, We Look Back at the March for Science

    Type
    Blog Post
    Published
    Oct. 24, 2017, 1:54 p.m.
    ... would not hide behind these two fingers the ninety-nine cubits of Aristotle, nor would you mention my...where our medicines, technologies, defense, and governing policies are developed primarily by wishful thinking...culminated in the current President holding and campaigning on many views that don't line up with the scientific... us. I worried the march would be a simple, unquestioning celebration of science, at a time when scientists...Jennifer Stancill I am a research biologist by training, so of course I felt compelled to participate ...say that I was supremely proud of Nashville that morning. Despite the rain, about 4,000 supporters of science...one person vocally protesting us, he was not threatening in any way. Participating in this march proved...

  47. Thoughts on the Future of Research 2015 Boston Symposium

    Type
    Blog Post
    Published
    Nov. 25, 2015, 4 p.m.
    ...postdoctoral pay/benefits commensurate with work training and experience, better align trainee expectations...temporary and defined period of mentored advanced training to enhance the professional skills and research...Associations around Boston - From Panel Session 2: Defining the Postdoc: An Institutional Perspective. This...of the academic research workforce, and we are beginning to identify potential solutions. Keynote speaker...structural problems seem to boil down to that fact that training in the academic research environment is setup ... is this very idea that postdocs are acquiring training to become professors that is provided as an explanation... they don’t make in wages, they make up for in training. Unfortunately, these low wages also make it so...

  48. Hot Plasmids - February 2022

    Type
    Blog Post
    Published
    Feb. 22, 2022, 2:15 p.m.
    ...imaging of cellular targets Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...frankenbody (FB) construction from the complementarity determining region of an anti-FLAG scFv and a stable scFv...small epitope tag (FLAG is eight amino acids, HA is nine), multiple repeats of the tag can recruit multiple...10.1038/s41467-019-10846-1.   Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...Gabrielle Clouse While many CRISPR-based genetic screening methods have primarily used Cas9 to knock out ...the Broad Institute have expanded the genetic screening toolkit by optimizing a Cas12a-based method for.... 2021 To validate this optimized genetic screening technique, researchers identified a synthetic ...

  49. A Deep Dive into BioBuilder

    Type
    Blog Post
    Published
    Oct. 11, 2017, 1:44 p.m.
    ...club of its kind, running from start to finish during the academic year and remaining affordable for all...through October 19. The BioBuilder Learning Lab The BioBuilder Learning Lab is a fully equipped, state-of-the-art...capitalize on the excitement found through experiential learning. “Students may forget the technical details they...handful of local high school teachers receiving training modules in synthetic biology has grown into a ...a project-based curriculum, BioBuilder expands learning outcomes by emphasizing the importance of critical...you can get involved with BioBuilder Details for joining BioBuilder’s programs can be found on its website...production of fire-resistant housing insulation, and cleaning up salt runoff from winter icing.BioBuilderClub...

  50. To Each HIS Own

    Type
    Blog Post
    Published
    March 2, 2023, 2:15 p.m.
    ...the tag and how many do I need? When designing a plasmid containing a polyhistidine tag, you’ll need to...single cell has tens of thousands of proteins functioning in a variety of ways. How do scientists focus...are immobilized on a bead or resin and a sample containing the protein of interest flowed through. The protein...the metal ions.      Fig. 1: A sample containing a polyhistidine tagged protein of interest (purple... (blue, green, orange) flows through the Ni2+-containing resin. The protein of interest (purple) binds...and is immobilized along with a polyhistidine containing contaminating protein (orange). A low imidazole...relatively low frequency amino acid, proteins containing two or more adjacent histidines are present in...
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