...short region of DNA: guide RNAs targeting different genes in an organism's genome, for example. Amplification...Molecular Biology Reference How do I process my Addgene pooled library? Introduction Please refer to our...depositor for a specific pooled library available at Addgene. We recommend following the depositor's specific...amplification. This protocol is designed to be as general as possible but note that individual libraries ...results. If you obtained the pooled library from Addgene, these protocols can be found on the Pooled Library...our protocol on Bacterial Transformation . The generalized pooled library amplification protocol is expected... a diagnostic digest and high-throughput next generation sequencing (NGS). Select a restriction enzyme...

... pHelper; pRC (Rep-Cap), plasmid expressing your gene of interest Triton X-100 Benzonase/DNAse I (Millipore... 0.45 μm polyethersulfone (PES) filter system, Nalgene, 565-0010 (or for larger volumes 1000 mL 0.45 µm... µm Rapid-Flow PES Filtration Unit, Nalgene 167-0045) Pro-Tip Do not use filters made of materials other...

...Blog Posts Addgene Protocols Viral Service Introduction This protocol describes how to generate a monoclonal...Selective pressure on this heterogeneous cell pool could lead to reduced transgene expression over time, as...more likely to retain stable transgene expression. Other methods of generating a monoclonal cell line include...for generating stable cell lines with lentivirus . Day 0: (optional) Seed cells for the generation of ...highest or lowest transgene expression ( Figure 2 ). Sample Data Figure 1: Generation of monoclonal cell...This protocol describes how to generate a monoclonal cell line from a polyclonal pool of stable cells...expressing clones take over the polyclonal cell pool. Generating a monoclonal cell line by limiting dilution results...

... Educational Resources Addgene Videos Addgene Videos Browse Addgene's collection of video content...using Addgene's resources, and career advice. For written lab protocol material, visit our Addgene Protocols...Storing and Handling Addgene Plasmids What to do after you receive your plasmids from Addgene Instructions for...samples from Addgene Technology Transfer How to Order A step-by-step walkthrough of Addgene's ordering process...this first installment of the Addgene Careers series, we sit down with Addgene Outreach Scientist Jessica...she loves about working at Addgene! Maria Soriano Maria Soriano, from Addgene's UK office, discusses her...A collection of Addgene's video content, including how-to screen captures, lab procedure protocols, and...

... master mix preparation and droplet generation (droplet generation BSC) to ensure no contamination of ...cap the PCR tubes. Generate the Droplets Bring the PCR tubes to the droplet generation BSC. Without disturbing...holder to the droplet generator. Close the lid and wait for the droplets to be generated. Once the droplets...12–5E+13 GC/mL, where GC is genome copies and generally represents the physical number of viral particles...surfaces with 10% bleach. Safety Warnings AAV is generally considered biosafety level 1 but may require BSL...Molecular Biology Grade Water, Hyclone, SH30538.02 GeneAmp 10X PCR Buffer, Applied Biosystems, N8080129 ddPCR...Pluronic F-68, Thermo Fisher, 24040032 Droplet generation oil, Bio-Rad, 1863005 DG8 cartridge, Bio-Rad,...

...using ready-to-use bio-safe Coomassie G-250 stain (Addgene uses SimplyBlue SafeStain) and ImageJ software....using ImageJ software and a standard curve is generated. The ratio of the antibody protein content to ...reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other vendors should...accommodate slight differences between products. Addgene does not endorse or recommend specific products... may not be functional. This sample would fail Addgene QC and would not be used. Section 3: Image Analysis...11469.296 4655.113 16642.522 16124.409 96.887% Pro-Tip Addgene only uses recombinant antibody preps that have ...

...reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other vendors should...accommodate slight differences between products. Addgene does not endorse or recommend specific products...intended to support reproducibility in science. General Considerations The ELISA can be completed in a ...The color will change from yellow to blue and generally takes between 15 to 30 min. High concentrations...spectrophotometer. Use the spectrophotometer to generate a standard curve . If you don’t see any color ...

...reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other vendors should...accommodate slight differences between products. Addgene does not endorse or recommend specific products... optimal antibody concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL of the diluted... optimal antibody concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL fluorescently-labeled...

...reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other vendors should...accommodate slight differences between products. Addgene does not endorse or recommend specific products...intended to support reproducibility in science. General Guidelines Acrylamide percentage varies in SDS-...depending on the size of your cell pellet but will generally be between 250–1000 µL . The ideal lysis buffer...

...designed by the Addgene nonprofit plasmid repository and is being shared outside of Addgene for informational...administrators, or anyone else associated with Addgene, can be held responsible for your use of the information...

...achieves this by using centrifugal force, which is generated by spinning the sample at a high speed. Being ...specific temperatures. This protocol will cover the general procedure and features to keep in mind when using... larger containers. Ultracentrifuges are also generally floor sized centrifuges that can hold larger containers...

...designed by the Addgene nonprofit plasmid repository and is being shared outside of Addgene for informational...administrators, or anyone else associated with Addgene, can be held responsible for your use of the information...

...Use this protocol to generate lentivirus...downstream applications such as stable-cell line generation. Workflow Timeline Day 0: Seed 293T packaging...adherent cells) 0.45 μm polyethersulfone filter, Nalgene, 565-0010 Microcentrifuge tubes, Neptune 3745.X...

...reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other vendors should...accommodate slight differences between products. Addgene does not endorse or recommend specific products...30 kDa MWCO buffer exchange column. (Optional) Regenerate the column by washing with 25 mL of Protein A...

...your DNA sample concentration and purity using Addgene’s DNA Quantification Protocol....ratio of nucleic acid to protein (260/280) is generally used as an indicator of the purity of DNA samples...

...annealed can be cloned directly into the overhangs generated by restriction digest of existing sites in the... additional bases to complement the overhangs generated when digesting the vector with EcoRI and SalI ...

...downstream applications. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that...adherent cells) 0.45 μm polyethersulfone filter, Nalgene, 565-0010 (for viral preps, if prep was not previously...

...consideration the information above, primers should generally have the following properties: Length of 18-24...

... medium. This dot is composed of millions of genetically identical bacteria that arose from a single bacterium...

...temperature between 4°C and room temperature. This general protocol suits almost any water bath. Video Watch...