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We narrowed to 1,053 results for: FER

Showing: 151 - 180 of 1053 results

  1. Genetic Code Expansion

    Type
    Collection
    ... directed evolution to charge the tRNA with a different amino acid. They are typically expressed from ...

  2. Trimmer Lab NeuroMab Collection

    Type
    Collection
    ...Plasmids scFv Plasmids Nanobody Plasmids Protocols References The UC Davis/NIH NeuroMab Facility (Link opens...NeuroMab Protocols (Link opens in a new window) References Andrews NP, et al. 2019. A toolbox of IgG subclass-switched...

  3. Molecular Biology Reference

    Type
    Guide
    ...Educational Resources Molecular Biology Reference Molecular Biology Reference Jump To: Molecular Biology Basics...contain a reporter gene (for example, luciferase or GFP) that offers a read-out of the activity of the genetic... C EDQVDPRLIDGK S Tag KETAAAKFERQHMDS SB1 PRPSNKRLQQ Webpage and Blog References Addgene's blog , including...Addgene's molecular biology reference guide, including information on molecular genetics, plasmids, sequencing...verification Resources Genetic Code Webpage and Blog References Protocols Origins of Molecular Genetics The concept... Joshua Lederberg coined the term plasmid, in reference to any extrachromosomal heritable determinant....bacterium to another by a type of horizontal gene transfer (conjugation), usually providing a benefit to ...

  4. 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...

  5. Lentiviral Vector Guide

    Type
    Guide
    ...packaging genes. However, they are different viruses and require different isoforms of these packaging components...second-generation transfer plasmids as well. Third-generation plasmids include (Figure 4): Transfer plasmid — ...vectors separate transfer, envelope, and packaging components of the virus between different plasmids. The...activate the promoter on the transfer plasmid. Many of the lentiviral transfer plasmids that have been deposited...genes. From an experimental standpoint, the main difference between lentiviruses and gamma-retroviruses is...three (or four, for third generation) plasmids: Transfer plasmid — contains transgene, sgRNA, or shRNA ...systems, due to a wide range of infectivity for different cell types (known as tropism). Lentiviral vectors...

  6. 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...

  7. 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...

  8. Antibody Guide

    Type
    Guide
    ... dates. Buffers Buffers are a concern for conjugating and signaling molecules, as some buffer components...heavy chain constant region” or Fc. Different isotypes play different roles in the immune response, and ...removed from the buffer before the reaction. However, it can be added back to buffers after the conjugation...of colors activated by different wavelengths, allowing for easy differentiation between multiple antibodies... In some cases, it may be preferable to buy small aliquots of many different antibodies and do batch validation...an antigen, while the specific binding site is referred to as an epitope. Production Antibodies are typically...typically produced in one of three different methods: in animals, in cultured immune cells, or through...

  9. Adeno-associated virus (AAV) Guide

    Type
    Guide
    ... via a different plasmid. In order to produce rAAV vectors, three plasmids are needed: Transfer plasmid...s Molecular Biology Reference . Production The three production plasmids (transfer, packaging, and helper...2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central...immune response. These features have made AAVs the preferred choice to be used as viral vectors for gene therapy...transgene, gRNA, or shRNA of interest into the transfer plasmid can be done with most standard cloning...restriction enzyme, Gibson Assembly, or Gateway. Some transfer plasmids may have limited restriction sites or...recombination of unstable regions, like the ITRs found in transfer plasmids. This will ensure that the repeats will...

  10. 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...

  11. Gamma-Retroviral Vector Guide

    Type
    Guide
    ...three different plasmids containing the transfer, envelope, or packaging components. The transfer plasmid...packaging genes. However, they are different viruses and require different isoforms of these packaging components...type. From an experimental standpoint, the main difference between lentiviruses and gamma-retroviruses is...gamma-retroviral vectors, you need three plasmids (Figure 2): Transfer plasmid — contains transgene, sgRNA, or shRNA ... gene, due to a wide range of infectivity for different cell types (known as tropism). Figure 2: Gamma-retroviral...transgene, gRNA, or shRNA of interest into the transfer plasmid can be done with most standard cloning...restriction enzyme, Gibson Assembly, or Gateway. Some transfer plasmids may have limited restriction sites or...

  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. Plan Your Experiment

    Type
    Guide
    ...many different Cas proteins available, and researchers can adapt these experiments for different organisms...Desired Genetic Manipulation Different genetic manipulations require different CRISPR components. Selecting...editor variants often have different optimal editing windows. Be sure to refer to the literature associated...discrepancies with the reference sequence you used for gRNA design. Addgene offers many plasmids containing...Activation/interference Needs good primer design Western blot Knockouts Activation/interference Needs a ...Expression and Delivery Validate Genetic Edit References CRISPR ( C lustered R egularly I nterspaced S...have expanded the CRISPR toolbox to include many different types of edits, including point mutations, base...

  17. 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...

  18. Adenovirus Guide

    Type
    Guide
    ... (A to G). Different serotypes exhibit varying tissue tropisms, meaning they preferentially infect certain...from different species. Used to alter infectivity and tropism. pShuttle Class of shuttle/transfer vectors.... These genes are now supplied in trans , via different plasmids or the packaging cell line, which not...need two plasmids: Shuttle plasmid (also known as transfer plasmid) — containing the transgene of interest...recombination between the transfected adenoviral transfer plasmid and the E1 region integrated in the packaging...vectors Third-generation rAdV vectors are also referred to as "gutless" or high capacity AdV (HCAdV) vectors...transgene, gRNA, or shRNA of interest into the transfer plasmid can be done with most standard cloning...

  19. 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)....

  20. 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...

  21. 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 ...

  22. 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...

  23. The Golden Stopwatch Award: Recognizing Facilitators of Research

    Type
    Blog Post
    ...your TTO have a question? Refer them to Addgene’s Tech Transfer Team: techtransfer@addgene.org Congratulations...Addgene Help Center: Tech Transfer Technology Transfer Information Tech Transfer News: A Reintroduction ...Addgene account for Tech Transfer Offices. They’re packed with tech transfer resources (summaries, electronic...stats and strategies with tech transfer offices. Drop us a line at techtransfer@addgene.org. Albert Einstein...requests from Addgene MTA: material transfer agreement Technology Transfer: the exchange of intellectual property...addgene.org identifies the hold-up. Your Material Transfer Agreement is stuck in “pending.” Fear not, there... to make MTA turnaround faster with technology transfer offices (TTOs). The latest of these efforts is...

  24. Five Popular Model Organisms

    Type
    Blog Post
    ...utilized mice to study how different mutations in leukemia impact different treatment regimens (Zuber ...example, the Stainier lab studied the proliferation and differentiation of β-cells in the the developing zebrafish...β-cells that originate in different regions of the pancreas and produce different levels of insulin (Hesselson...mouse (Mus musculus) Let’s start with the most preferred mammalian model organism of many researchers: ...injecting mice with retroviral vectors that harbored different mutations found in leukemia, the Lowe lab was ... gene expression in specific cells of the fly (Pfeiffer et al., 2010). Yeast (Saccharomyces cerevisiae...at a time. Zebrafish embryos are also laid and fertilized externally, allowing scientists to easily manipulate...

  25. Deep Dive: Statistical Tests (Comparisons)

    Type
    Blog Post
    ...test assesses the sum of differences between pairs). Samples arising from different subjects or populations...test assesses the difference of the group means). Unpaired t-tests can also be referred to as two-sample...significant differences between the means of your groups. However, it will only tell you if differences exist...samples are different from each other, or if every permutation of pairs within the five is different. To identify...groups to determine if they are statistically different. They are classic parametric tests and one of ... whether or not two samples are statistically different than one another, a two-tailed test should be ...control group. If you only care if one group is different in a certain direction (i.e., you just want to...

  26. CRISPR Guide

    Type
    Guide
    ...Alternatives Resources Plan Your Experiment Web References PAM Sequences Glossary Publications CRISPR Overview...techniques . Collectively, these techniques are referred to as CRISPR ( C lustered R egularly I nterspaced...measure of fidelity, so sometimes they will be referred to as increased fidelity enzymes. hfCas9s were...pyrimidine-to-pyrimidine) Base transversions - edit to different classification of nucleotide base (purine-to-pyrimidine...opposite strands Prime editing and base editing both offer high efficiency and low indel formation compared...genetic changes, including moving entire genes to different locations, by combining other gene editing mechanisms...DNA-targeting of CRISPR-Cas systems. You may also see them referred to as INTEGRATE ( I nsertion of T ransposable ...

  27. Antibodies 101: Beyond Surface Labeling

    Type
    Blog Post
    ..., Addgene uses 'labeling' to refer to antibodies and 'staining' to refer to small molecule dyes. Indirect...first fixed and then permeabilized, using two different buffer solutions. Fixing causes protein cross-linking...nucleus, a different fix/perm kit is required (to be precise, the fixing solution is different while the...plot (Figure 4b). Through this method, differences in proliferation between cell populations can be visualized...divisions).   Figure 4: Proliferation dyes can visualize proliferative capacity and speed of cells...lineage markers, like many of the cluster of differentiation (CD) proteins found on immune cells. However...use 'labeling' and 'staining' interchangeably to refer to any technique that combines a marker with a signal...

  28. Starter guide to induced pluripotent stem cells (iPSCs) part 1:  A renaissance in regenerative medicine

    Type
    Blog Post
    ...maintaining the undifferentiated “stem” state and, Their potency or capability to differentiate into specialized...special types of cells that can develop or “differentiate” into more specialized cells with specific functions...function adequately [1]. Stem cells’ ability to differentiate into multiple cell types makes them useful models... cell has the ability to produce all of the differentiated cells in an organism, including the embryonic...Pluripotency: When a cell has the ability to differentiate into cells of any of the three germ layers: ...detail below [2]. Multipotency: When a cell can differentiate into multiple cell types of the same lineage...name suggests, cells under this category can differentiate into only one cell type. Muscle satellite cells...

  29. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    ... virus can be stored at 4 °C and buffer exchanged the next day. Refer to the AAV Iodixanol Gradient Ultracentrifugation... AAV packaging plasmids. The cells need three different plasmids to produce AAV: 1) the RepCap plasmid...help mediate AAV viral replication; and 3) the transfer plasmid which encodes a transgene of interest ...more details. Pro tips I’m not going to lie, AAV transfer plasmids are kind of a pain to work with. These...structures that are prone to deletion from the transfer plasmid. The good news is there’s a few simple...to grow multiple bacterial cultures of your AAV transfer plasmid at 30 °C instead of 37 °C and then screen...restriction sites). Another way is to transform AAV transfer plasmids into bacterial strains, like NEB Stable...

  30. Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection

    Type
    Blog Post
    ...created phages. Transfer of this DNA to another cell is referred to as transduction. Transferred DNA once inside...donor can transfer genetic material to the recipient bacterium. The genetic material transferred is commonly...Horizontal gene transfer (HGT) is the movement of genetic material between organisms. It plays a key ...unique to the lab. Let’s take a look at these different methods of DNA insertion. Transformation Transformation...Various factors promote natural transformation in different bacteria such as growth phase of the cells (Baltrus...plasmid, the genes on the plasmid can give them different phenotypes. For example, this plasmid encodes ...cell. Once inside, phages can follow one of two different life cycles: lytic or lysogenic. Lytic phages ...
Showing: 151 - 180 of 1053 results