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We narrowed to 1,045 results for: fer

Showing: 161 - 180 of 1045 results

  1. Chemogenetics Guide

    Type
    Guide
    ...light-emitting luciferase and a light-sensing optogenetic element. When the luciferase substrate (luciferin) is ...efficiencies. Depending on the type of luciferase used, the luciferin substrate may be the native coelenterazine...activity in neurons LMO Luciferase Opsin Ligand Effect Outcome (in neurons) Reference LMO3 sbGLuc VChR1 CTZ...RASSLs DREADDs PSAMs LMOs Planning Your Experiment References Chemogenetic tools are actuators for specific... of chemogenetic receptors. There are several different types of DREADDs that can be broadly classified...inhibitory KORD DREADD was developed to respond to a different ligand, Salvinorin B (SALB). Scientists have shown...Receptor Effector Ligand Effect Outcome (in neurons) Reference Rq(R165L) Human M3 muscarinic Arrestin-2/-3 CNO...

  2. Optogenetics Guide

    Type
    Guide
    ...Introduction Microbial Opsins Optical Switches References Introduction The field of optogenetics integrates...naturally occurring microbial opsins that respond to different wavelengths of light, like blue or yellow light...light. These various opsins also initiate different electrochemical responses, such as nonspecific cation... blue light activated cation channel. ChR2 is preferred over ChR1 because ChR2 has higher conductance ...optimization, and chimeric fusion of domains from two different ChRs. These ChR variants still function as light-gated...Color of activation light. There are a variety of different activation wavelengths, ranging from blue to yellow...rendering the experimental procedure less invasive. Different activation wavelengths also make it possible to...

  3. Sequencing Primers

    Type
    Guide
    ... Plasmid Reference Molecular Biology Reference Sequencing Primers Sequencing Primers...convenience. For reference information, please consult Addgene's Molecular Biology Reference Page . All listed...AGTCAAGTAACAACCGCGA 3' end of luciferase, forward primer LucNrev CCTTATGCAGTTGCTCTCC 5' end of luciferase, reverse primer...sequences listed on the left are provided for your reference. Addgene does not distribute primers. For sequencing... primer LucNrev CCTTATGCAGTTGCTCTCC 5' end of luciferase, reverse primer M13 Reverse CAGGAAACAGCTATGAC...GGGCTGGCAAGCCACGTTTGGTG 3' end of glutathione-S-transferase, forward primer SP6 ATTTAGGTGACACTATAG SP6 promoter...GGGCTGGCAAGCCACGTTTGGTG 3' end of glutathione-S-transferase, forward primer pGP704-R AACAAGCCAGGGATGTAACG...

  4. Plan Your Experiment

    Type
    Guide
    ...Desired Genetic Manipulation Different genetic manipulations require different CRISPR components. Selecting...be present in a single lentiviral transfer vector or separate transfer vectors May contain reporter gene...experimental goal, you are ready to start navigating the different reagents that are available for your particular...particular cell type or organism without a specific preferred mutation Cas9 (or Cas9 nickase) Single (or dual...can make a limited set of mutations. Repress or Interfere (CRISPRi) Reduce expression of a particular gene...targeting promoter elements of target gene Many different activators exist, including the multi-plasmid ...transduction CRISPR elements are inserted into an AAV transfer vector and used to generate AAV particles (for...

  5. Promoters

    Type
    Guide
    ... Plasmid Reference Molecular Biology Reference Promoters Promoters Definition A promoter is...Addgene's molecular biology reference material covering promoters, transcription and the transcription...mRNA transcript produced. The antisense strand is referred to as the template strand or non-coding strand...transcription of DNA into mRNA. Promoter binding is very different in bacteria compared to eukaryotes. In bacteria...promoter region. Each sigma factor recognizes different core promoter sequences. Operons Although bacterial...regulation, like operons. Operons are a cluster of different genes that are controlled by a single promoter... types of RNA polymerases that all transcribe different genes. RNA polymerase I transcribes genes encoding...

  6. Cloning

    Type
    Guide
    ...you to do many different molecular biology techniques (such as fusing it with different tags, putting ...Addgene's reference guide on molecular cloning techniques, including information on seven commonly used... Molecular Biology Reference Choosing a Molecular Cloning Technique Molecular Cloning...availability of starting materials or just personal preference. Check out our blog on choosing the right cloning...Gateway® destination plasmids have been made with different promoters, tags and fluorescent proteins. Thus... variety of promoters and into backbones with different selection cassettes). Although gateway cloning...

  7. Guide to Using Pooled Libraries

    Type
    Guide
    ...pooled libraries are single preparations of many different plasmids. Plasmids within a given library have...the same backbone, but they express or target different genes. Some libraries cover the majority of the... library from the Moffat lab has over 175,000 different gRNA-containing plasmids). Amplifying and Using...screen. If you need to amplify the library, please refer to the depositor’s protocol for the best results...conducting pooled library screens. Addgene does not offer next-generation sequencing services. For an NGS ...Genome-Scale, Knockout Screening Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional Screens...

  8. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    ...optimizing your transfer buffer.  Adjusting the buffer For wet and semi-dry transfers, you can adjust ...methanol concentration in transfer buffer is < 20% Wet transfer Increase transfer time Increase voltage...decrease alcohol in transfer buffer Small proteins: Over-transferring Transfer with two membranes...increase alcohol in transfer buffer Mixed proteins: Transferring issues Transfer with two membranes...be under-transferring.  Membrane pore size Membranes come in different pore sizes for different proteins...large.  Transfer method   Wet transfers are more efficient, so if you’re struggling to transfer a large...consider using a wet transfer with a long transfer time. Be aware that the transfer works by electricity...

  9. Technical Design of a Western Blot

    Type
    Blog Post
    ...There are many different lysis buffers available commercially, and most common buffers have recipes available...Types of transfers Your transfer options are wet, semi-dry, or dry/electroblot. A wet transfer is the most...  Note that each type of transfer requires different equipment. Wet transfers can be done in an insert... while semi-dry transfers require a semi-dry transfer cell. Dry/electroblot transfers only work in the...is typically sufficient.  Lysis buffer When choosing your lysis buffer, consider the subcellular location...and Tris gels run proteins in different patterns, due to their differing chemistries. You therefore cannot... 12% 10–250 kDa Different sizes 4–12% gradient 4–250 kDa Different sizes 4–20% gradient...

  10. Deep Dive: qPCR

    Type
    Blog Post
    ...melting profiles (Dwight, 2011).  Reference Genes  Choosing a set of reference genes deserves more attention...attention than it usually receives. Different reference genes have shown to have as much as sixfold variation...Passive Reference Dyes Whatever your pipetting method, some variation will occur. A passive reference dye,...Liu and Saint No Raw Data Reference and target genes can have different efficacies DART-PCR No ...will soon become a qPCR master. References and Resources  References Biassoni R; Raso A. (eds.), Quantitative...mRNA, which can be used as a proxy measure for differences in protein expression. (If you're just getting...assume that qPCR can accurately detect expression differences of twofold or greater with good statistical significance...

  11. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    ... wet transfer system the sandwich is completely submerged in a tank filled with a transfer buffer formulated...systems efficiently transfer a range of protein sizes in minutes without any transfer buffer. These systems...electric current. The wet transfer process is very effective in transferring a broad range of protein ...overnight.  In contrast, a semi-dry transfer only requires enough buffer to saturate the sandwich and typically...validated for immunoblotting. Different immunoassays detect proteins in different states. For example, in immunoprecipitation... other antibody topics. Resources and references References Aydin S (2015) A short history, principles...western, proteins are: (1) separated by size, (2) transferred to a membrane, and  (3) detected using antibodies...

  12. Custom CRISPR Screens & the Green Listed Software

    Type
    Blog Post
    ...heterogeneous population, cells behaving differently (e.g. different growth properties, migration abilities... modified cells are transferred into an animal and the behavior of the transferred cells is followed. ...targeting all differentially expressed genes that are known drug targets, or all differentially expressed ...best gRNAs by the depositing lab. The different labs use different algorithms to calculate which would ...were not found in the reference library. It’s not uncommon that genes have different names, and the software...generation sequencing to identify which gRNAs are differentially expressed in the separate groups and, as a ...is not targeted by only one gRNA, instead many different gRNAs are used for each gene. At the moment, 13...

  13. Selecting Your Plasmid Purification Kit

    Type
    Blog Post
    ...Addgene blog Plasmid Preps: Different Purity, Different Quantities, Different Uses Plasmids 101: Optimizing...lysis buffer ratio and result in incomplete lysis. You may be able to scale the amount of buffers used ...TakaraBio) offer yeast-plasmid specific kits.  Medium/High-throughput – Many manufacturers offer bead- and...quantity.   Figure 1: Plasmid preps fall into different categories based on their relative qualities and...fairly self explanatory and achieved by performing different sized preps, such as miniprep, midiprep, etc. ...what do we mean by DNA quality? There are a few different issues that can arise during the plasmid purification...which can happen if you leave the sample in a lysis buffer for too long or if you get a little overzealous...

  14. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    ...stable expression of the transgene   Transfer plasmids The transfer plasmid is the vector you clone your...the control of two different promoters. Both the single and dual promoter transfer vectors can be used...genetically modify primary cells. Amongst the different vectors used, lentiviral vector constructs have...background Until the development of viral vectors, gene transfer into mammalian cells often relied on chemical ...These techniques are still used and efficiently transfer plasmid DNA efficiently into dividing immortalized...lines but reach their limits when it comes to transferring DNA into primary cells or non-dividing cells... particle (Gag, Pol, Tat, Rev and Env) and the transfer plasmid encoding the insert of interest as well...

  15. Your Lentiviral Plasmid FAQs Answered

    Type
    Blog Post
    .... While these differences may seem subtle they result in a key physiological difference between lenti-...3rd-generation transfer vectors apart? A3: Addgene defines 2nd- or 3rd-generation transfer vectors based...packaging systems differ between 2nd- and 3rd-generation vectors? What does this mean for transfer vectors? A4...protein and consequently are considered safer. Second-generation transfer plasmids MUST be packaged with a 2nd-generation... Are transfer plasmids replication competent? What is SIN? A6: Most (if not all) Addgene transfer plasmids...which is rarely more than 30% of the transfer plasmid. Many transfer vectors are SIN. Packaging plasmid...ingredients: the lentiviral packaging vector, a transfer vector including the gene of interest, and the...

  16. Mobile-CRISPRi: Bringing CRISPRi to Diverse Bacteria

    Type
    Blog Post
    ...Mobile-CRISPRi: a modular approach for CRISPRi transfer Mobile-CRISPRi offers two advantages over existing bacterial...chromosomal copy of the RP4 transfer machinery mobilize these plasmids for transfer to recipient cells during...disease. However, the rate of transfer was variable. They measured transfer rates by comparing the number... P. mirabilis, had only ~1 transfer per 1,000,000 colonies. Higher transfer rates are better suited for...Mobile-CRISPRi plasmids at Addgene! References Auchtung, Jennifer M., et al. "Biology of ICEBs1, an integrative...them. For example, gene knockdown with CRISPR interference (CRISPRi) has been limited to lab-adapted bacteria...diverse bacteria species. Existing protocols can transfer CRISPRi into a single bacterial strain, such as...

  17. Antibody Validation for Flow Cytometry

    Type
    Blog Post
    ...approach is RNA interference (RNAi). The most common method is transfection of short interfering RNA (siRNA...For CD365, they examined two different antibody clones produced by different vendors. The specific epitope... panels, where different manufacturers may have the same clone available with different conjugates. Summary...commercial antibody manufacturers. References and resources References Ayoubi, R., Ryan, J., Biddle, M. ...include a discussion of the Human Leucocyte Differentiation Antigens (HCDM) workshop approach, which focuses...one size fits all. The approaches used will be different depending on the sample and target type. To start... For intracellular targets, we usually test 3 different fix/perms (methanol, PFA-saponin, PFA-triton)....

  18. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ..., A mutant Renilla luciferase, catalyzes the oxidation of its substrate, luciferin, which releases energy... neomycin or luciferase) to create a final lentiviral vector containing up to 5 different elements. Lentiviral...bioluminescence resonance energy transfer (BRET), whereby photons emitted by a Renilla luciferase variant are used to...alternative to the widely used luciferase reporter gene plasmid pGL3 pGL3 luciferase reporter gene plasmids... issues with fluorescent pH sensors, a set of luciferase-fluorophore pH fusion reporters, pRSETb-pHlash...pHlash, that use Bioluminescence Resonance Energy Transfer (BRET) were developed by the Johnson Lab . Characteristics...energy as a photon. The photon energy is transferred to the acceptor fluorophore, a circularly permuted...

  19. Multiplexed Overexpression of Regulatory Factors (MORF) collection

    Type
    Blog Post
    ... directed differentiation To understand how each human TF can contribute to differentiation, the Zhang...atlas. Computational analysis was used to infer differentiation trajectories and identify both up- and down-regulated... scientist at [email protected]! References and Resources  References Joung J. et al., A transcription...patterns determine everything from stem cell differentiation fate to tumor suppression (and most everything...construct. Cells were cultured following a differentiation protocol before performing single-cell RNA ...embryonic stem cells. Transduced stem cells were differentiated and single cell RNA-seq was performed to map...    This atlas linked TF expression to differentiation outcomes as well as global transcriptional ...

  20. Plasmids 101: Codon usage bias

    Type
    Blog Post
    ...most organisms on Earth, but different organisms have different preferences for the codons they use to ... organisms have different codon usage biases? The reasons for varied codon preferences among organisms...poorly charged tRNAs, different regions of the protein may be translated at different rates. The ribosome...Organisms often need to express genes at different levels under different conditions. With varied codon usage...constraints have molded which codons are used preferentially in which organisms - organisms have codon usage...your organism of choice. Unfortunately, codon preferences make it so you cannot choose among the possible... evolutionary constraints that lead to these preferences and what can we do about them? Read on to find...
Showing: 161 - 180 of 1045 results