...your Gene Selection of suitable 21-mer targets in your gene is the first step toward efficient gene silencing...unrelated genes. TIP: Addgene recommends that you select multiple target sequences for each gene. Some sequences...directed against 15,000 human and 15,000 mouse genes. Addgene is working with the TRC to make this shRNA ... human and mouse genes. Protocols...B.1 Determine the optimal 21-mer targets in your gene B.2 Order oligos compatible with pLKO.1 C. Cloning...expression of puromycin. Puro R Puromycin resistance gene for selection of pLKO.1 plasmid in mammalian cells...origin of replication. Amp R Ampicillin resistance gene for selection of pLKO.1 plasmid in bacterial cells...

...resistance gene to effectively create an extra part, one part has half of the antibiotic gene and the adjacent...have at least the correctly assembled antibiotic gene. This trick can also enable replacement of "inverse...change in a plasmid (such as point mutations). Generate DNA segments by PCR. Run PCR product on an agarose...

...verification of important plasmid features (such as the gene/insert, fusion proteins, point mutations, deletions... help you verify the ends of the insert. Longer genes may require internal primers that anneal to a specific...charge. What primers were used by Addgene during quality control? Addgene lists the primers used to obtain...and need to design a custom primer, Addgene recommends using Addgene's sequencing results as a reference...wish to contact Addgene about the accuracy of your plasmid, please email help@addgene.org and include: The...regions of your plasmid that you wish to confirm. Addgene's plasmid information pages recommend 5’ and 3’ ...specific region of the insert. You can find Addgene's sequencing results by clicking on the "View Sequences...

...containing plasmids differing in their resistance genes, as one does not need to prepare separate batches...plasmid containing the carbenicillin (amp) resistance gene (or other antibiotic resistance) Procedure Day 1...plate). Please note we have found that there is generally a broad range of antibiotic concentrations that...

...Plasmids can carry one or more antibiotic resistance genes, which confer resistance to a specific antibiotic...carrying them. The presence of an antibiotic resistance gene on a plasmids allows researchers to easily isolate...

...novel methods improves infectious titer and yield." Gene therapy 6.6 (1999): 973-985 (Link opens in a new...Felix D., Rist Wolfgang, and Lamla Thorsten. Human Gene Therapy Methods. August 2015, 26(4): 147-157. doi...

...targeting human and mouse genes. Virus Name Description Link to Video General Transfection Introduce plasmid...Cells Produce adeno-associated virus to deliver your gene of to the target of your choice AAV Purification...A wide variety of protocols from Addgene that can be used for basic molecular biology, plasmid cloning... Addgene Protocols On this page you'll find practical lab...column. You can find even more video content on the Addgene Video Page . We hope that you'll find these protocols...own work. Please feel free to email us at help@addgene.org with any questions. Intro to the Lab Bench Introductory...restriction digest and ligation Cloning by PCR Generate restriction sites by PCR Modification by Annealed...

...recombinant plasmid is connecting the insert DNA (gene or fragment of interest) into a compatibly digested...1 will get you in the ballpark for average size genes and vectors, this ratio is really meant to refer...reactions involve DNA fragments that have been generated by restriction enzyme digestion . Most restriction...depicts the ligation of two sticky ends that were generated by EcoRI digestion: Usually, scientists select...

...Plasmids can carry one or more antibiotic resistance genes, which confer resistance to a specific antibiotic...carrying them. The presence of an antibiotic resistance gene on a plasmid allows researchers to easily isolate... by a plasmid carrying the antibiotic resistance gene. The following protocol will allow you to make your...

...resistance gene for use as a selectable marker in bacteria. Scientists have made many genetic modifications...containing the correct antibiotic. The resistance gene on your plasmid must match the antibiotic on the... chemical or electrical based transformation, generating what are commonly referred to as 'competent cells...This outgrowth step allows the bacteria time to generate the antibiotic resistance proteins encoded in ...

...Transfections allow for transient expression of a gene of interest in a target cell line and can be useful... Protocols General Transfection General Transfection You may also like... Viral...Service Introduction This protocol describes a general method for transfecting mammalian cells using linear...

...assume that you are beginning a new project on your gene of interest (YGOI for short). You might need to ...'t want to express the antisense version of your gene!) Are in frame with tags or fusion proteins in the...Choosing enzymes) Many DNA analysis tools, including Addgene’s Sequence Analyzer , allow you to identify which...

...Protocol to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector...This protocol can be used to generate stable cell lines expressing a gene of interest from an integrated... Protocols Generating Stable Cell Lines with Lentivirus Generating Stable Cell Lines with Lentivirus... may have 1 copy of the transgene, while others have >1 copy of the transgene in their genome. This can...high levels of the transgene may have reduced growth rates, especially if that transgene is toxic. Eventually...low-level transgene expressors may take over the culture. To overcome this, consider generating monoclonal...observed using transient transfection approaches, generating cell lines using lentiviral vectors enables long-term...

...concentrated plasmid DNA. This page will discuss the general procedure for purifying plasmid DNA from bacterial...streak a plate to get individual colonies and to generate liquid bacterial cultures , please see those pages...μL. The protocol below is meant to describe the general procedure for purifying plasmid DNA from bacterial... TE buffer 3 M Na-acetate (pH 4.8) Protocol: Generalized DNA Purification Grow an overnight culture of...

... vector that is better suited for analyzing the gene’s function. The process is shown graphically in the...which we are adding EcoRI and NotI sites to Your Gene of Interest (YGOI) for ligation into a recipient..., you should use a DNA analysis tool, such as Addgene’s Sequence Analyzer , to allow you to identify which... site to improve cutting efficiency. You can generally add any 6 bases, but you should ensure that the... 18bp of homology to the ORF). We now need to generate the reverse-complement of this sequence so that...that we can successfully amplify the ORF. You can generate the reverse-complement using existing software...

... viruses per cellular genome: Since the cellular gene RPP30 is used as a reference to the cellular genome...Titer by a Novel Droplet Digital PCR Method. Hum Gene Ther Methods . 2018 Apr;29(2):96-103. doi: 10.1089...holder to the droplet generator. Close the lid and wait for the droplets to be generated. Once the droplets...Fluorescence Titration Assay AAV Titration by qPCR Addgene Viral Service Lentivirus Guide Virus Blog Posts...1E+09 TU/mL, where TU is transducing units and generally represents the number of infectious viral particles...with 10% bleach. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that...Centrifuge, Thermo Scientific, 10199-452 Reagents GeneJet Genomic DNA Purification Kit, Thermo Fisher, K0721...

...manufacturer's suggested leader sequence fused to your gene of interest, in frame with the start codon or tag...an example of LIC experimental design. Search Addgene's collection of empty LIC cloning vectors Protocol...transformation, following a standard protocol . Search Addgene's collection of empty LIC cloning vectors...

...a single, short region of a DNA molecule (single gene perhaps) to be copied multiple times by Taq Polymerase...cations can also be used for PCR-mediated DNA mutagenesis. A higher cation concentration increases the ...

...Transfections allow for transient expression of a gene of interest in cell culture. This protocol describes...Antibodies Blog Posts Molecular Biology Reference General Transfection Protocol Recombinant Antibody Purification...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 For antibody-expressing plasmids...

...-derived inverted terminal repeat sequences. Hum Gene Ther Methods. 2012 Feb;23(1):18-28. (Link opens ...molecules/μL to generate a standard curve: One option is to use plasmid #59462 from Addgene. The values highlighted...Calculation Size in bp of Addgene plasmid #59462 : 6208 bp Concentration of Addgene plasmid #59462 : 1.07 ...AAV prep using qPCR, SYBR Green Technology, and Addgene’s AAV Titration protocol. Protocols...Microcentrifuge tubes 96-well optical plate Pipette tips General Considerations Always run standards and samples...titer >3 x 10 13 GC/mL, use dilutions 5–8 Note: at Addgene, samples typically range from 1 x 10 12 GC/mL to...