... Addgene Protocols On this page you'll find practical lab protocols that you can use for ...Plasmid Cloning Protocols for constructing and analyzing your plasmids. Virus Protocols for titering and...A wide variety of protocols from Addgene that can be used for basic molecular biology, plasmid cloning...range of applications, with videos for select protocols in the right-hand column. You can find even more...Addgene Video Page . We hope that you'll find these protocols useful in your own work. Please feel free to email...started in the lab. Basic Molecular Biology These protocols are the building blocks for many more complicated...and testing your virus preparations. Antibodies Protocols for common antibody applications. Intro to the...

...Protocol, tips, and FAQ for how to perform a restriction digest of plasmid DNA... Protocols Restriction ...diagnostic digest . Last Upload: Oct. 11th, 2016 Protocol Video Watch the video below to learn how to analyze...

...based cloning. Protocol: Gel Purification Follow the Agarose Gel Electrophoresis Protocol with the following...Protocol for how to purify DNA from an agarose gel, including tips to help improve the resolution and... Protocols Purifying DNA from an Agarose Gel Purifying DNA from an Agarose Gel ...

...video protocols, tips on using Addgene's resources, and career advice. For written lab protocol material...questions. Protocols Video protocol guides for standard laboratory procedures. How-To Videos Screencapture...including how-to screen captures, lab procedure protocols, and career advice videos. Educational...material, visit our Addgene Protocols page . Please feel free to email us at [email protected] with any questions...career insight and advice from real Addgenies! Protocols (Link opens in a new window) Video Link Description...culture bacteria in the lab Pouring LB Agar Plates Protocol Streaking Bacteria on Plates Isolate single bacterial... on an agar plate Streaking Bacteria on Plates Protocol Diagnostic Restriction Digest Verify a plasmid...

...Procedure Option #1 - Solution D Protocol Before starting this protocol, make sure to prepare solution ...and storing those in -80°C. Solution D Protocol TRIzol® Protocol Top...Follow this protocol to perform RNA extraction without a kit. Protocols... Protocols RNA Extraction Without a Kit ... Molecular Biology Reference Introduction This protocol describes how to extract RNA from tissues or cells...cells without a kit. The general steps of this protocol include homogenization/lysis of cells or tissues... of RNA, precipitation, and resuspension. This protocol provides two options: using Solution D (see reagent...

...Antibodies Immunocytochemistry Protocol Western Blot Protocol Introduction This protocol describes how to affinity...outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted ...This protocol describes how to affinity purify recombinant antibodies from cell culture supernatant using... Protocols Recombinant Antibody Affinity Purification with Protein... believe that sharing the full details of our protocols supports reproducibility and accelerates science...waste procedures. Last Update: February 7, 2022 Protocol Video Watch this instructional video to learn ...compatible with 50 mL conical tubes NanoDrop spectrophotometer 37 °C, 5% CO 2 incubator with shaking platform...

...specific protocol if one is available. If a pooled library does not yet have an amplification protocol, the...the following protocol can be used as a starting point for CRISPR libraries. This protocol allows the amplification...these protocols can be found on the Pooled Library page for the specific library. This protocol assumes... our protocol on Bacterial Transformation . The generalized pooled library amplification protocol is expected...Follow this protocol to perform amplification of CRISPR pooled plasmid libraries in Escherichia coli ... Protocols CRISPR Library Amplification CRISPR Library Amplification You may also like... Pooled...pooled library material pages for amplification protocols that have been developed by the depositor for ...

...Immunocytochemistry Protocol Recombinant Antibody Purification Protocol Antibody Guide Introduction This protocol describes...outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted ...of each well at 450 nm on the spectrophotometer. Use the spectrophotometer to generate a standard curve...This protocol describes how to run an indirect ELISA against a purified antigen to demonstrate the expected... Protocols Antibody Validation Using the Indirect ELISA Method Antibody...purified antigen to validate an antibody. This protocol will describe how to create a standard curve using... believe that sharing the full details of our protocols supports reproducibility and accelerates science...

... Virus Blog Posts Addgene Protocols Viral Service Introduction This protocol describes how to generate...Workflow Timeline This protocol begins with a stable cell pool. For details, see our protocol for generating ...This protocol describes how to generate a monoclonal cell line from a polyclonal pool of stable cells... Protocols Cloning by Limiting Dilution Isolating a Monoclonal Cell Population by Limiting Dilution... at 4 °C. Polyclonal stable cell pool: see our protocol for generating stable cell lines with lentivirus...to grow under the conditions described in this protocol. Conditioned medium (details below) can be used...monitored regularly for transgene expression. This protocol has been optimized for adherent cells but can ...

... Protocols Centrifugation Centrifugation Protocol Intro to the Lab Bench Check...Check out more protocols and videos to help you get started in the lab! Introduction A centrifuge is a ...or keep samples at specific temperatures. This protocol will cover the general procedure and features ... will have essentially the same components. The photo to the left shows a front view of an open tabletop...tabletop microcentrifuge with the lid open, the rotor with microfuge tubes in place, and the control panel...centrifuge, with tubes evenly distributed around the rotor. How to Centrifuge Now that you know the different...working smoothly. Place your sample tubes into the rotor so that they are distributed evenly around the center...

... Guides Virus Blog Posts Mol Bio Protocols Introduction This protocol can be used to purify AAV of any...a new window) from Sigma-Aldrich. Protocol Video Watch the protocol video below to learn setup and use...Follow this protocol using Iodixanol gradient ultracentrifugation to get pure adeno-associated virus ... Protocols AAV Purification by Iodixanol Gradient Ultracentrifugation...purified AAV in vivo . The iodixanol gradient in this protocol is composed of steps that separate out contaminants... one (long) day. Equipment Ultracentrifuge T70i rotor or equivalent Reagents Opti-Prep (60% Iodixanol)... Centrifuge at 350,000 x g for 90 min in a T70i rotor at 10 °C. Pro-Tip If you need more time, you can...

...from a bacterial culture, including protocols, tips, and FAQ... Protocols Recovering Plasmid DNA from Bacterial...concentrations ranging from 150 ng/μL to several μg/μL. The protocol below is meant to describe the general procedure...purification without using a kit, you can find a protocol for kit-free plasmid mini-prep at the bottom of... 70% ethanol TE buffer 3 M Na-acetate (pH 4.8) Protocol: Generalized DNA Purification Grow an overnight...bacteria . Pro-Tip Refer to appropriate DNA prep protocol for volume of bacteria to grow (low copy plasmids... DNA concentration and purity click here . Top Protocol: Kit-free Alkaline Lysis Plasmid Miniprep Prepare...

...purity. This can be done with a spectrophotometer. A spectrophotometer uses the absorbance/transmission... instructions for the spectrophotometer specific to your lab. Spectrophotometer Tips Before measuring ...purity using Addgene’s DNA Quantification Protocol. Protocols... Protocols DNA Quantification DNA Quantification You...labs have a NanoDrop, which is a very small spectrophotometer that can accurately read DNA concentration...measuring any samples, be sure to ‘blank’ the spectrophotometer using the solution the DNA is resuspended in, ...NanoDrop. It is a good idea to re-zero any spectrophotometer between samples if they are expected to vary...

...Guide Immunocytochemistry Protocol Recombinant Antibody Purification Protocol Introduction Western blot...outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted ...This protocol describes the basic steps for lysing cells, determining total protein concentration in ... Protocols Western Blot Western Blot You may also like......antibodies specific to the protein of interest. This protocol describes the basic steps for lysing cells, determining... believe that sharing the full details of our protocols supports reproducibility and accelerates science...immunoblotting low molecular weight proteins. This protocol uses a dry transfer device but can be adapted ...

...Antibody Guide Western Blot Protocol Recombinant Antibody Purification Protocol Introduction Immunocytochemistry...outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted ...This protocol describes the basic steps for fixing and staining cells in culture with a primary antibody... Protocols Immunocytochemistry Immunocytochemistry You may...protein and a fluorescent secondary antibody. This protocol outlines the steps for fixing and labeling HeLa...protein using the formaldehyde fixation method. The protocol may need to be optimized for different cells, ... believe that sharing the full details of our protocols supports reproducibility and accelerates science...

...Reference General Transfection Protocol Recombinant Antibody Purification Protocol Introduction Transfections...outcomes when using these protocols. However, please be aware that your protocol may need to be adjusted...Lab protocol for transfection of HEK293 cells to produce recombinant antibody. Protocols... Protocols Transfection...expression of a gene of interest in cell culture. This protocol describes how to transfect suspension HEK293 cells... believe that sharing the full details of our protocols supports reproducibility and accelerates science...supernatant (see our Recombinant Antibody Purification Protocol ). Store the supernatant at 4 °C until ready to...

... Virus Blog Posts Mol Bio Protocols Viral Service Introduction This protocol can be used to titer lentiviral...Protocol that can be used to titer lentiviral preparations carrying a fluorescence marker. Protocols... Protocols ...of infection (MOI) in downstream studies. This protocol was developed for 293T cells but can be adapted...lines and selection markers. Note, because this protocol uses microscopy to determine the percentage of...and lots of FBS to find one suitable with your protocols. FBS can be purchased already heat-inactivated...complete containing 10 μg/mL polybrene. Note, this protocol was developed using low titer lentiviral vectors...

... lid and rotors should be loaded and unloaded in a biosafety cabinet. BSL-2 safety protocols require bloodborne... Protocols Lab Safety Lab Safety for Biosafety Levels One... BSL-2) Intro to the Lab Bench Check out more protocols and videos to help you get started in the lab!...Introduction There are four biosafety levels. This protocol provides information for both biosafety level ...may be some times when you may be working with a protocol that requires shaking or mixing, which may produce...addition to BSL-2 guidelines below, including PPE protocols . Working in a BSL-2 laboratory requires laboratory... simple, following appropriate BSL-1 and BSL-2 protocol goes a long way. It is the easiest way to ensure...

... Virus Blog Posts Mol Bio Protocols Viral Service Introduction This protocol describes a general method...Use this protocol to transfect mammalian cells with your plasmid of interest. Protocols... Protocols General Transfection... of protein function. We specifically use this protocol with a subclone of HEK293T cells optimized for...and lots of FBS to find one suitable with your protocols. FBS can be purchased already head inactivated...µg of DNA into 500 µL of Opti-Pro SFM. Pro-Tip Endotoxins can inhibit transfection; therefore, plasmid ...plasmid DNA purification should include an endotoxin removal step. For high quality plasmid DNA, the plasmid...

...like... Viral Vector Guides Mol Bio Protocols Introduction This protocol can be used to produce lentivirus...Use this protocol to generate lentivirus Protocols... Protocols Lentivirus Production Lentivirus Production You may...cells using a polyethylenimine (PEI) transfection protocol. This procedure can be modified for alternative...and lots of FBS to find one suitable with your protocols. FBS can be purchased already heat inactivated... optimized for each transfer plasmid. Pro-Tip Endotoxins can inhibit transfection; therefore, plasmid ...plasmid DNA purification should include an endotoxin removal step. For high quality plasmid DNA, the plasmids...