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...aspirate, using a new aspirating pipette for each well. Detach cells by incubating with 200 µL TrypLE for 1...concentration of each sample on a spectrophotometer. Prepare 25 ng/µL stocks of each sample. Samples can...use. Wipe down all pipettes and surfaces with 10% bleach. Safety Warnings Lentivirus is generally considered...magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) Trypsin EDTA (TrypLE, Thermo... of 80-fold dilution 200 µL 400 µL Add 150 µL of each viral dilution to a well of a 6-well plate (1 dilution...Mix the cell suspension well before seeding. Mix each well with a 1 mL pipette 5–10 times. The final volume...plates. Be sure to use a new aspirating pipette for each well as to not contaminate cell conditions with ...

...the cells have detached from the plate and can be isolated. When trypsinized, detached cells will exhibit...magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) Trypsin EDTA (TrypLE, Thermo...the transgene, and the specific conditions used. Each 96-well plate described in this protocol could, ...on the A549 cells described in our sample data , each 96-well plate gave rise to 15–20 monoclonal lines...cells this is about 2 × 10 6 cells in a 10 cm dish. Each 10 cm dish should be seeded in 10 mL DMEM complete...Prepare approximately 10 mL of this cell solution for each 96-well plate you plan to seed. If you choose not... Transfer 100 µL of the 5 cells/mL solution into each well of a 96-well plate. By doing this, you are ...

... µL of 4X sample buffer to each sample. Add 2 µL 10X reducing agent to each sample. Spin the sample briefly...peaks representing each protein band. Use the line tool to connect the bottom of each peak. Select the ...µL of 0.25 mg/mL with 10 µL PBS Transfer 5 µL of each standard to a microfuge tube. Add 8 µL of PBS to...gel. Carefully remove the comb from the gel. Rinse each well with 200 µL 1X MOPS running buffer. Fill the...skip one lane before loading samples. Load 20 µL of each recombinant antibody sample to the appropriate well...gel easier. Cover the electrophoresis chamber and attach to a power supply. Run the gel at 150 V for 1 h...Select Image . Select Type . Choose 8-bit . Determine each lane of the gel as follows: Figure 2 Using the box...

...the first three wells of each row, using a different standard solution for each row of a 96-well ELISA ...multichannel pipette to add 200 µL wash buffer to each well. Washing can be done by hand but if available...multichannel pipette, add 200 µL of blocking buffer to each well. Cover the plate with a plate seal and incubate...multichannel pipette, add 100 µL of secondary antibody to each well. Cover the plate with a plate seal and incubate...

...knockout approaches. In these cases, we would recommend a knockdown approach instead. In this approach, you...using one or more of the approaches outlined below. Knockout cell line approach Genetic knockout-based...Knockdown approach For cell types where knockout studies are not feasible, an alternative approach is RNA... the target of interest. This approach maps onto the orthogonal approach within the five pillars and is...knockout/ or knockdown approach should be preferred. The overexpression approach is therefore best suited... use of orthogonal approaches, cell treatments, and independent antibody approaches in different assay...Wright, A. K. A., Sousa, J., Weston, C. A., Chachi, L., Roach, K. M., & Bradding, P. (2019). Validation ...

...497 Add 0.5 mL of a single viral dilution to each well (each well gets one dilution, so a 6-well plate will...50,000 cells) into each well of the 6-well dish. This brings the total volume in each well up to 1.5 mL...expression observed using transient transfection approaches, generating cell lines using lentiviral vectors...magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) 0.45 μm polyethersulfone filter...reverse transduction" by seeding 50,000 cells into each well of the 6-well dish. These cells will be added...polybrene, the final concentration of polybrene in each well should be 10 µg/mL. Pro-Tip Transducing too...antibiotic, which may not be stable at 37 °C. To achieve a stable cell pool, the antibiotic selection should...

... 96-well plate: Load 5 μL of each standard in duplicate Load 5 μL of each sample in duplicate. Do not ...well by pipetting back and forth multiple times at each step Reagent Preparation Master Mix: Count the number...that there is enough master mix for all samples). Each sample requires 15 μL of master mix. Pro-Tips Use...standard curve is obtained, make a small aliquot of each standard (enough for 1 or 2 plates) and store at...use within 1 week. Keep track of the Ct value for each standard over time. They should remain within 0.5...carrier DNA to a final concentration of 4 ug/mL to each standard dilution. Dilute DNase-treated and AAV ... dilution series is critical. Make sure to pipet each dilution up and down at least 10 times, and use ...

...minutes at 95°C in a PCR machine or in a beaker of boiling water. If using a PCR machine, incubate the sample...recommends that you select multiple target sequences for each gene. Some sequences will be more effective than... for Inserts Day 1: 1. Inoculate 5 colonies from each ligation into LB + 100 μg/mL ampicillin or carbenicillin...different amounts of virus as needed. Day 1: a. For each plasmid to be transfected, plate 7×105 HEK-293T ... NOT use polystyrene tubes), make a cocktail for each transfection: 1 μg pLKO.1 shRNA plasmid 750 ng psPAX2...amount of Fugene® and OPTI-MEM necessary given that each reaction will require 6 μL FuGENE® + 74 μL OPTI-...temperature. e. Add 80 μL of FuGENE® master mix to each tube from step c for a total volume of 100 μL. Pipette...

...use. Wipe down all pipettes and surfaces with 10% bleach. Safety Warnings AAV is generally considered biosafety...place on ice. Wipe down a DG8 cartridge holder with bleach and place in the Biological Safety Cabinet (BSC... a single channel 1–10 µL pipette to add 5 µL of each viral sample to Dilution 1 in the 48-well dilution...avoid generating aerosols. Be sure to change tips each time. Dilution 1 (20X): 5 µL in 95 µL 1X PCR buffer...well freezer block. Add 20 µL of the master mix to each PCR tube. Be careful to dispense to the bottom of...to get to temperature. Once the temperature is reached, place the PCR plate with the foil onto the metal...the following equation to calculate the titer for each sample: $$T = {R*C*D*1000\over V}$$ Where: T = Titer...

...store multiple types of tube for each plasmid we have at Addgene. Each sample at Addgene has four layers...image below). Each plate has numbers and letters to give coordinates, from A1 through H12. Each plate is also...media with antibiotics at each stage. The barcodes and LIMS help us track each sample, but we also have...? Different shelves? Different racks on each shelf? Give each container a specific location to know exactly... Addgene’s lab, a series of events is triggered. Each step is tracked through barcoded tubes that are ...Software    The plasmid's journey begins First, each package is opened and the contents are carefully...have been transported across oceans and continents. Each deposit may consist of anywhere from one to hundreds...

... Other variants of Spinach include Baby Spinach, a miniaturized version of Spinach that retains similar... Hammond’s group created Spinach2 using site-directed mutagenesis of Spinach. In doing so, they identified...was consequently brighter than Spinach. The initial studies using Spinach2 showed its ability to track RNA...imaging. Bunch of baby spinach, or BoBS, contains tandem repeats of Baby Spinach. It was developed in a...twice as bright as Spinach2 when bound to DFHBI. It’s also smaller than any form of Spinach - only 49 nucleotides...Addgene’s collection. Green fluorescent aptamers Spinach and Broccoli and their derivatives are aptamers... and non toxic to cells. Because it rapidly photobleaches, DFHBI can only be used for qualitative, but...

...: 3325793 Schumacher MA (2012) Bacterial plasmid partition machinery: a minimalist approach to survival...control plasmid replication of each plasmid using unique replication machinery. However, when plasmids have...NTPase (Schumacher, 2012).  During cell division, CBPs bind to the centromere-like region on each plasmid... partitioning each type of plasmid into a separate daughter cells (Figure 2) (Ebersbach et al., 2005)....origin of replication and the replication control machinery (Need a refresher on the Origin of Replication...they compete for the same replication control machinery within the cell. Most plasmids encode a negative...through inhibiting translation of replication machinery proteins, or directly inhibit replication by binding...

...a fluorophore will bleach. This is, however, not the full story. The photobleaching rate does not depend... 2-fold does not reduce photobleaching by half. How exactly the photobleaching rate depends on power is...experiments. As the photobleaching rate is not linearly related to power, the conclusions reached at high power...thanks to our guest bloggers Joachim Goedhart and Marieke Mastop! Joachim Goedhart is an assistant professor...This post was contributed by guest bloggers Joachim Goedhart and Marieke Mastop from the Section of Molecular...previous post in this series described a practical approach to selecting a bright fluorescent protein. In ...to select a photostable fluorescent protein. Photobleaching is the irreversible destruction of a fluorophore...

.... This is the direct approach to immunoimaging assays. But if the direct approach isn’t giving you enough...indirectly, with each protein of interest.    Figure 1: The direct approach (left) versus the...almost always use the indirect approach, while flow cytometry’s standard approach is the direct method. ELISAs...immunocytochemistry may all use either the direct approach or indirect approach, depending on the requirements of the...this is much easier said than done! Each technique, and sometimes each individual assay, will need to be...interest, it may be time to look at the indirect approach - which means it’s time to talk about secondary...binds to the primary antibody. In the indirect approach, the signaling molecule is conjugated to the secondary...

...-charge from the ABRC Outreach website. Seeds, which are free for K-12 teachers and available for a nominal...support. Growing capacity - ABRC outreach today Over the years, the outreach efforts of ABRC have grown. A...In 2017 alone, ABRC staff reached more than 4,000 people through our outreach efforts. Our education partnerships... you a biology teacher or professor? Whether you are looking to develop a new teaching resource or you... guest blogger Courtney Price, the Education & Outreach Specialist for the Arabidopsis Biological Resource...samples to researchers and educators in 60 countries each year. Introducing Arabidopsis Arabidopsis is an ...interested in studying animals than plants, and biology teachers’ preference for using animal systems to demonstrate...

...when our middle school’s 8th grade science teachers reached out to their students’ STEM-oriented parents...presentation five times to groups of about 50 students each, and each group went on its own wonderful tangents. Some...about how I could combine all of these roles by reaching out to my school district not just as a dad, but...presentations. If you’re a scientist thinking about reaching out to your community’s schools, here are a few...technology into the school curriculum. Our Parent Teacher Association (PTA) has been instrumental in supplementing... means to request funding for events, consider reaching out to it. PTAs have more flexibility than the...math tables!  Be realistic about what you can teach  5-10 year olds in a single evening. Grades K-5 ...

...students and teachers can investigate together. Teachers decide which BioBuilder lessons to teach, and how.... A summer teacher professional development program that trains nearly 100 teachers each year, with workshops...needs of students and teachers. It’s possible to join a half-day workshop for teachers, to schedule a three-day...synthetic biology into the hands of students and teachers in the U.S. and beyond. “I want to learn more ... a question more and more students are asking. Teachers are also asking, “How do I engage my students .... Built primarily from her 20 years of college teaching experience, Kuldell sought to create a program...Educational Foundation in partnership with high school teachers to boost interest and understanding around the...

...Starting Wipe down all pipettes and equipment with 10% bleach prior to use. Warm the affinity column, binding...gently pour off the ethanol storage buffer. Tightly attach LabMate PD10 Buffer Reservoirs to the top of the...columns, refer to the manufacturer’s instructions. Attach the Gravitrap column to a clamp on a clamp stand...s to mix. Determine the protein concentration of each fraction on the NanoDrop Spectrophotometer using... 3x , discarding buffer from the collection tube each time. Place column in a new 50 mL LoBind collection...NanoDrop Spectrophotometer to see if the sample has reached the desired concentration. If the sample concentration...until the volume of the sample is reduced enough to reach the desired concentration. Pro-Tip Periodically ...

...explore genome editing approaches for exon skipping. Addgene depositor Charles Gersbach used paired zinc finger... respectively. Although each study used slightly different methods, they each observed the same positive...CRISPR-mediated exon skipping approach in mice, DMD researchers are very excited. This approach may also be applicable...so researchers have set their sights on another approach. Since shorter forms of dystrophin can still be...for postnatal genome editing in mice. Could this approach work with dystrophin exon skipping? For such a...Long et al., Nelson et al., and Tabebordbar et al. each chose to try exon skipping in DMD mice, which have...-type levels. With regard to off-target effects, each group finds very low to no off-target activity at...

...membrane and press each gently down onto a plate of colonies. Mark the orientation of each membrane on its... thick filter paper and saturate each with lysis solution (~3 ml each), pouring off any excess. When your... excess liquid from each membrane and press a fresh Kimwipe down firmly onto each to remove cell debris...Many thanks to our guest blogger Jessica Sacher! Jessica Sacher is a microbiology PhD student at the University... post was contributed by guest blogger, Jessica Sacher, a microbiology PhD student at the University of...interesting, much of what a phage faces as it approaches a host cell surface are complex polysaccharide...description of a technique published by Simpson, Sacher, and Szymanski in January 2016 in Viruses (5). ...