...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...

...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)....

..., 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...

... 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 ...

...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...

...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...

...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...

...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...

..., 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...

...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...

... 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...

...pseudotype γ-retrovirus using different envelopes to modify tropism. Briefly, different envelope plasmids can ...engineered γ-retroviruses differ from lentiviruses? There are a few important differences between engineered ...packaging; however, they are different viruses and thus use slightly different isoforms of these packaging...Vectors 101 eBook, 1st edition.  References and resources References Swift S, Lorens J, Achacoso P, Nolan...following components are needed: γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA ...sequences, which facilitate integration of the transfer plasmid sequences into the host genome. Typically... the host genome upon viral transduction. Most transfer plasmids available from Addgene were derived from...

...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 ...

...Tris-EDTA (TE), just Tris buffer, or some other variations. Does it make a difference? Short answer: yes. This...choices: TE, Tris buffer, or water Figure 2: Tris-EDTA, Tris, or water? The buffer you choose could...TE (10 mM Tris-HCl,1 mM EDTA, pH 8.0) buffer is the best buffer for preserving the stability of your preparation...can be Tris buffer (with no EDTA) or water. These are two of the choices that companies offer for the elution...preparation can be eluted in water or in your buffer of preference, and it will be stable for years. Plasmid... preparation for a long time. Tris buffer controls the pH, while the EDTA chelates any divalent cations...through their kit. The advantage of using a Tris buffer is that it allows better control of the pH preparation...

...through horizontal (or “lateral”) gene transfer. Horizontal gene transfer can occur naturally through transformation...conjugation (direct transfer from one cell to another). Bacteria need special fertility genes to be capable...Emily P. Bentley in October 2024. References and Resources References Demanèche, S., Bertolla, F., Buret...Here at Addgene, we often refer to plasmids as lab or experimental tools. They certainly are very handy... evidence that lightning can also promote gene transfer in soil bacteria! (Demanèche et al., 2001) Beneficial...humans only noticed last century! Nevertheless, the transfer of plasmids carrying these genes between bacteria...Historically, colicin-producing plasmids have been referred to as “Col plasmids” or “Col factors.” Virulence...

...host range is determined by the different hosts that a plasmid can transfer to, replicate in, and persist...stable, can be easily cloned in different bacteria, and can be transferred to various experimental hosts... that can replicate in different bacteria and express proteins within different host organisms. pBBR1-...recombinant vectors at Addgene!   References and resources References Doran, K. S., Konieczny, I., & Helinski...host range, identify a few of the features that confer broad host range in plasmids in nature, and provide... they carry among organisms belonging to many different phylogenetic groups (Jain and Srivastava 2013)...be used by scientists across disciplines.  What confers broad host range in plasmids? Not all plasmids ...

...your poster Check conference guidelines To begin, you should always follow conference rules when designing...useful to use two different font styles; one for your section titles, and a different one for the body ...distinguished. The UCLA Library offers some Powerpoint based templates with different column numbers for a good... the most of your conference attendance Read all of our blog posts about conferences .... But, before you can show off your work at a conference, you need to first make your poster. While a ...understand that presenting your work in poster format differs in many ways from writing a manuscript. If you ...you have ever attended a scientific conference, or even a lab recruiting session at your university, you...

...not transfer precisely evenly across the entire blot. So how can you accurately compare different lanes...assuming that all differences in the loading control are due to loading and transfer errors — not to any...reflect massive differences in protein abundance. Validating the assumptions for different loading controls...detection range be linear! Resources and References References Aldridge, G. M., Podrebarac, D. M., Greenough...errors like uneven pipetting or irregular membrane transfer. Figure 1: A loading control is a type ...Researchers use later steps in this chain (blue) to infer or calculate things about earlier ones. In practice...relationship poses problems for proteins of very different abundance, consider the following example gel....

...There are many different flavors of MTAs, but the Uniform Biological Material Transfer Agreement (UBMTA...process that differs.  When you deposit a plasmid with Addgene, your organization’s tech transfer office is...for more information. Happy transferring!    Many thanks to our Tech Transfer team members Maura Coughlin...Addgene, then you’ve been involved in the material transfer agreement (MTA) process. Even though this process...curious about the hows and whys of the material transfer agreement, read on!  What is the MTA?  An MTA...UBMTA) is one of the most commonly used ones for transfers between academic institutions.  What is the UBMTA...the creation of a standardized agreement on the transfer of biological materials in 1995: the UBMTA. Because...

... deposited by Promega here.    References and resources   References Allikalt, A., Purkayastha, N., Flad... scientists. Seeing inside these specimens has offered illumination on biochemical processes that are ...Figure created with biorender.com. Fluorescence refers to the emission of light by a substance (fluorophore...energy due to non-radiative energy losses. This difference in emission results in a slightly longer wavelength...phenomenon is known as the Stokes Shift, or the difference in wavelength between peak excitation spectrum...vary in their chemical structure, leading to differences in their excitation and emission wavelengths,...target specific organelles like mitochondria, and different dyes can result in varying cytoplasmic background...