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... 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...

...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...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...

...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...

...Recombinant Antibody Purification Protocol Introduction This protocol describes how to determine the purity...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... believe that sharing the full details of our protocols supports reproducibility and accelerates science...ImageJ (Link opens in a new window) or a similar photo software to determine the relative intensity of ...

...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...

... this protocol to perform digital droplet PCR (ddPCR) for lentivirus vector titration.... Protocols ddPCR...Lentivirus Guide Virus Blog Posts Introduction This protocol describes how to use droplet digital PCR (ddPCR...ddPCR) to titer lentivirus vectors. This protocol specifically uses primers and probes targeting integrated...normalization. The dilution series outlined in this protocol is based on an LV titer range of 1E+05–1E+09 TU...dilutions depending on their particular sample. This protocol was modified from the publication Wang et al. ...Determine the concentration of each sample on a spectrophotometer. Prepare 25 ng/µL stocks of each sample. Samples...

... 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 ...

... 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...

...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...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...various confluencies. This AAV production protocol should be started with cells that are ~80% confluent (center...

... 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... 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...

... Protocols Personal Protective Equipment (PPE) for BSL-1 ...-2 Labs 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 ...Conclusion Although simple, following appropriate PPE protocol goes a long way. It is the easiest way to ensure...