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We narrowed to 976 results for: nin

Showing: 161 - 180 of 976 results

  1. Protocols for Molecular Biology, Plasmid Cloning, and Viral Preps

    Type
    Protocol
    ...Plasmid Cloning Name Description (Link opens in a new window) Link to Video Restriction Cloning Assemble...ligation Cloning by PCR Generate restriction sites by PCR Modification by Annealed Oligo Cloning Add a short...single reaction Ligation Independent Cloning (LIC) Scarless cloning with Type II restriction enzymes and...and T4 polymerase pLKO.1 - TRC Cloning Vector Cloning protocols for using the pLKO.1 vector, a backbone...can be used for basic molecular biology, plasmid cloning, and titering and testing your viral preparations... for many more complicated procedures. Plasmid Cloning Protocols for constructing and analyzing your plasmids...How to Design a Primer Key considerations when designing primers Watch the Video! Sequence Analysis Verify...

  2. Plasmids 101: Common Lab E. coli Strains

    Type
    Blog Post
    ...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...

  3. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    ...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...

  4. How to Be an Excellent Trainee

    Type
    Blog Post
    ...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...

  5. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ...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...

  6. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ...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...

  7. Pushing the Limits of DNA Assembly

    Type
    Blog Post
    ...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...

  8. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...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...

  9. Plasmids 101: Plasmid Incompatibility

    Type
    Blog Post
    ....  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 ...

  10. Plasmids 101: Colony PCR

    Type
    Blog Post
    ...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...

  11. Which Fluorescence Microscopy Technique is Best for Me?

    Type
    Blog Post
    ...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...

  12. CRISPR 101: Any Base Transversion Editors

    Type
    Blog Post
    ...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...

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

    Type
    Blog Post
    ... 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, ...

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

    Type
    Blog Post
    ...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...

  15. RANbodies: Reporter Nanobody Fusions

    Type
    Blog Post
    ...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...

  16. New Optogenetic Tools for Cytoskeleton and Membrane Control

    Type
    Blog Post
    ...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...

  17. Technical Design of a Western Blot

    Type
    Blog Post
    ... 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...

  18. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    ...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...

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

    Type
    Blog Post
    ...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-...

  20. Antibodies 101: Choosing the Right Isotype

    Type
    Blog Post
    ...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...
Showing: 161 - 180 of 976 results