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...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...

...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. (Link opens in a new window...including how-to screen captures, lab procedure protocols, and career advice videos. Educational...Resources Addgene Videos Addgene Videos Protocols How-To Videos Career Videos Browse Addgene's collection of...material, visit our Addgene Protocols page . Please feel free to email us at [email protected] with any questions...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...

...Antibody Guide Western Blot Protocol Recombinant Antibody Purification Protocol Introduction Immunocytochemistry...antibody against a target protein and a fluorescent secondary antibody. This protocol outlines the steps for...HeLa cells for a target protein using the formaldehyde fixation method. The protocol may need to be optimized...outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted ... expresses the protein, such as cells transfected with a plasmid to express the protein of interest, and...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. Protocols...

...Recombinant Antibody Purification Protocol Introduction This protocol describes how to determine the purity...generated. The ratio of the antibody protein content to the total protein content of the sample is determined...outcomes when using these protocols. However, please be aware that your protocol may need to be adjusted...Lab protocol to determine purity and concentration of recombinant antibodies using ready-to-use biosafe... Protocols Coomassie Purity Stain of Recombinant Antibodies Coomassie...concentration. After staining with Coomassie blue, protein band intensities are measured using ImageJ software... believe that sharing the full details of our protocols supports reproducibility and accelerates science...

...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 ...

...Guide 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...1000X protease inhibitor cocktail 25 mg Leupeptin 50 mg Antipain 250 mg Benzamidine 25 mL Aprotinin saline...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...

... 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...contaminants in each fraction. * AAV capsid proteins VP1, VP2, VP3; M protein marker. Image adapted from Strobel...Follow this protocol using Iodixanol gradient ultracentrifugation to get pure adeno-associated virus ... Protocols AAV Purification by Iodixanol Gradient Ultracentrifugation...purification and isolation of cells, organelles, lipoproteins, and macromolecules. Importantly, iodixanol ...purified AAV in vivo . The iodixanol gradient in this protocol is composed of steps that separate out contaminants...

... 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 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 ...between different types of hazards and to provide protective measures for each level, as each level has different...Equipment Lab coat Close-toed shoes Gloves Eye protection or face shields Eye wash Safety shower Sharps... entering the lab, put on the proper personal protective equipment (PPE) and wear it the whole time you...may be some times when you may be working with a protocol that requires shaking or mixing, which may produce...

...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 ...purified Desmin (blue) or a negative control protein, Apolipoprotein L3 (red) overnight, blocked, and incubated...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 Mol Bio Protocols Viral Service Introduction This protocol describes a general method...for short term studies of protein function. We specifically use this protocol with a subclone of HEK293T...Use this protocol to transfect mammalian cells with your plasmid of interest. Protocols... Protocols General Transfection...and lots of FBS to find one suitable with your protocols. FBS can be purchased already head inactivated...Incubate the cells 24–48 h before checking for protein expression. Sample Data Legend: Lenti-X 293T cells...be seen here by the amount of green fluorescent protein expression (green in the right panels) with a limited...

... 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...

...September 14, 2016 Protocol Video Before you begin this protocol, watch the protocol video below to get...like... Plasmids 101 Blog Posts Mol Bio Protocols Cloning Protocols Introduction Plasmids can carry one or...Use this protocol to prepare LB agar plates with antibiotic in your lab. Protocols... Protocols Pouring LB Agar Plates... the antibiotic resistance gene. The following protocol will allow you to make your own LB/agar plates...

...Protocol for Ligation Independent Cloning... Protocols Ligation Independent Cloning Ligation Independent...Methods Empty Backbone Guide Molecular Biology Protocols Overview Ligation Independent Cloning (LIC) obviates...Addgene's collection of empty LIC cloning vectors Protocol Step 1: Design Your Primers Primer design for ...counter-selection marker (see our restriction digest protocol ). After the digest is complete, you will need...all other nucleotides from standard polymerase protocol), causing the enzyme to perform exonuclease activity... for each transformation, following a standard protocol . Search Addgene's collection of empty LIC cloning...

... 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 ...Microfuge tubes or other suitable container Personal protective equipment (PPE) appropriate for the biosafety...and temperature, if applicable) listed in your protocol. Pro-Tip Spin speed is often given as Relative...Centrifuges are important tools for many different protocols. Improper use of a centrifuge can damage the equipment...

...Follow this protocol to produce adeno-associated virus (AAV) in HEK293 cells.... Protocols AAV Production... Molecular Biology Reference Introduction This protocol can be used to produce AAV from one Five Chambers...meter Stir plate Magnetic stir bar Sonicator Ear protection Vortex Reagents Adherent HEK293T cells (ideally...and lots of FBS to find one suitable for your protocols. FBS can be purchased already heat-inactivated...Other transfection reagents may be used in this protocol, but their conditions must be optimized. Plasmids...at 4 °C before proceeding with the purification protocol. Sample Data Figure 1: HEK293T cells at various...

... Virus Blog Posts Mol Bio Protocols Viral Service Introduction This protocol can be used to titer lentivirus...This protocol can be used to titer lentiviral preparations that confer antibiotic resistance. Protocols...of infection (MOI) in downstream studies. This protocol was developed for A549 cells but can be adapted...and lots of FBS to find one suitable with your protocols. FBS can be purchased already heat-inactivated...subjected to multiple freeze-thaw cycles. This protocol outlines the seeding of the cells at the same ...

... on size by gel electrophoresis . Protocol Video Watch the protocol video below to learn how to perform... of Polymerase Chain Reaction with protocol, tips and FAQ Protocols... Protocols Polymerase Chain Reaction (PCR) Polymerase...minutes at 72°C: A final extension to fill-in any protruding ends of the newly synthesized strands. Materials... Primer Design and PCR Design Primers. See our protocol on how to design primers . Note: (Link opens in...primers for detailed instructions and check out our protocol on plasmid cloning by PCR to learn how to design...

... Virus Blog Posts Mol Bio Protocols Introduction The AAV Titration protocol can be used to determine the... Technology, and Addgene’s AAV Titration protocol. Protocols... Protocols AAV Titration by qPCR Using SYBR Green Technology... an AAV prep using SYBR green technology. This protocol was validated using an internal reference AAV ... to those reported by these institutions. This protocol is for a 96-well plate with 20 μL reaction volume...sample to the bottom of the tube. Run the following protocol in your qPCR instrument using SYBR detection: ...

...Follow this protocol to perform digital droplet PCR (ddPCR) for purified AAV titration.... Protocols ddPCR Titration... Molecular Biology Reference Introduction This protocol describes how to use droplet digital PCR (ddPCR...recombinant Adeno-associated viral vectors (AAV). This protocol specifically uses primers and probes targeting... targets. The dilution series outlined in this protocol are based on an AAV titer range of 5E+12–5E+13...blog post on ddPCR for AAV quantification. This protocol was modified from the publication Lock et al. ...pre-chilled 96-well freezer blocks during use. In this protocol, a dilution series is prepared for each AAV sample...