... Protocols Fluorescence Titering Assay Fluorescence Titering Assay ...resulting in 25% fluorescent cells, then the titer is: (150,000 cells x 0.25 fluorescent x 100)/(1.5 mL)...resulted in 25% fluorescent cells, then the titer is: (150,000 cells x 0.25 fluorescent)/(0.015 mL) = ...preparations carrying a fluorescence marker. Protocols...to titer lentiviral preparations that carry a fluorescence marker. Determining the titer of your lentiviral...uses microscopy to determine the percentage of fluorescent cells, these cells can still be used in downstream...media, replace with fresh media Day 4–5: Count fluorescent cells Equipment Class II, Type A2 Biological ...

...This protocol describes the basic steps for fixing and staining cells in culture with a primary antibody...antibody against a target protein and a fluorescent secondary antibody....antibodies to detect antigens in cells. Here we describe the basic steps for fixing and labeling cells ...primary antibody against a target protein and a fluorescent secondary antibody. This protocol outlines the... tips and pipettes Rocking platform Tweezers Fluorescent microscope 0.5–10 µL single channel pipette 2...500 µL PBS on a rocking platform. Dilute the fluorescently-labeled secondary antibody to the desired concentration...generally ranges from 1-10 µg/mL. Add 500 µL fluorescently-labeled secondary antibody to the wells and ...

...transfection protocol Fluorescence Titering Assay for Lentivirus Quantify virus using fluorescence measurements...antibody applications. Intro to the Lab Bench Name Description (Link opens in a new window) Link to Video Personal...materials on a balance Basic Molecular Biology Name Description (Link opens in a new window) Link to Video Making...without a commercial kit Plasmid Cloning Name Description (Link opens in a new window) Link to Video Restriction... targeting human and mouse genes. Virus Name Description Link to Video General Transfection Introduce ... your lentivirus preparation Antibodies Name Description Link to Video Transfection Introduce plasmid ...

...This protocol describes the basic steps for lysing cells, determining total protein concentration in ...specific to the protein of interest. This protocol describes the basic steps for lysing cells, determining ...Nonfat milk powder 96-well microtiter plate Chemiluminescence substrate Plastic wrap Primary antibody Secondary... at RT on a shaking platform. Prepare the chemiluminescence substrate by mixing 1:1 reagent A to reagent...reagent B. Gently incubate the membrane in the chemiluminescence reagent for 5 min at RT . Cover the membrane...with clear plastic and use a gel imager with chemiluminescence detection or a dark room to detect the bands...

...This protocol describes how to generate a monoclonal cell line from a polyclonal pool of stable cells...Protocols Viral Service Introduction This protocol describes how to generate a monoclonal cell line from a ...type will be able to grow under the conditions described in this protocol. Conditioned medium (details ... specific conditions used. Each 96-well plate described in this protocol could, in theory, give rise to...protocol was performed at Addgene on the A549 cells described in our sample data , each 96-well plate gave rise...from Feng Zhang (Addgene plasmid #52962 ) and is described in Improved vectors and genome-wide libraries ...

...amplification of CRISPR pooled plasmid libraries in Escherichia coli cells....amplification of a pooled-plasmid library in Escherichia coli cells. Pooled libraries contain tens to ...activating pulse and follow all specifications described in the equipment manual. Immediately add 1 mL ...µl aliquots of cell/DNA mixture used (8 in the described protocol) using a new vented falcon tube for every...different concentrations of DNA. In our hands fluorescent dye methods tend to report lower values than ...

...Protocols Viral Service Introduction This protocol describes a general method for transfecting mammalian cells...with fresh media Day 3 or more (am): Observe fluorescence, harvest cells, or perform your experiment Equipment...batch of PEI is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the ...efficiencies as can be seen here by the amount of green fluorescent protein expression (green in the right panels...

...Protocols (Link opens in a new window) Video Link Description Related Page Making LB Agar Plates Create plates...Videos (Link opens in a new window) Video Link Description Related Page MTA Guide - Requests Your guide ...Videos (Link opens in a new window) Video Link Description Jessica Welch, PhD In this first installment ...

...This protocol describes how to run an indirect ELISA against a purified antigen to demonstrate the expected...Protocol Antibody Guide Introduction This protocol describes how to run an indirect Enzyme-Linked ImmunoSorbent...antigen to validate an antibody. This protocol will describe how to create a standard curve using known concentrations...

... Colony Formation Titration Assay Lentivirus Fluorescence Titration Assay AAV Titration by qPCR Addgene...Guide Virus Blog Posts Introduction This protocol describes how to use droplet digital PCR (ddPCR) to titer...and titrations Reference Wang Y, Bergelson S, Feschenko M. Determination of Lentiviral Infectious Titer...

... Bacterial Culture Introduction This protocol describes methodology for plating antibiotic over-agar for...transformation protocol. Plate 50 µL of transformed E. coli /rescue media suspension onto the agar and gently spread...

....9kb stuffer, visit www.addgene.org/10878/ . Description Vector Element U6 Human U6 promoter drives RNA...the pLKO.1 TRC-cloning vector. Addgene Plasmid Description pLKO.1 – TRC control Negative control vector ...license or rights of access or use to the materials described in the literature. The distribution of materials...

...bath on and set the appropriate temperature as described in your protocol. How to set the temperature will...water bath even if you are using disinfectants as described above in step 3. You will also need to maintain...

...This protocol describes how to affinity purify recombinant antibodies from cell culture supernatant using...Western Blot Protocol Introduction This protocol describes how to affinity purify recombinant antibodies ...

...batch of PEI is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the ...

...gene of interest in cell culture. This protocol describes how to transfect suspension HEK293 cells with ...

... several μg/μL. The protocol below is meant to describe the general procedure for purifying plasmid DNA...

...Description of Polymerase Chain Reaction with protocol, tips and FAQ...

...from Feng Zhang (Addgene plasmid #52962 ) and is described in Improved vectors and genome-wide libraries ...

...typically ROX dye), to normalize non-PCR–related fluorescence fluctuations and to minimize well-to-well variability...