...Transducing Cells Thaw the virus on ice. Prepare the following dilution series of virus in DMEM complete: Prepare...the first virus dilution, add 160 µL of viral stock to 240 µL of media. When adding the virus to the diluent...the following virus dilutions were used to transduce cells: Initial dilution factor of virus stock Dilution...titer, TU/mL V = Viruses per genome C = # Cells per well D = Dilution factor v = Virus volume, mL *In the...Titration by qPCR Addgene Viral Service Lentivirus Guide Virus Blog Posts Introduction This protocol describes...diluent, pipette 10 times, to remove virus from pipette tip. To mix the dilution, set the P200 pipette...as indicated in the chart below. Dilution Factor Virus Volume to Dilute Media Volume Total Volume 2.5 160...

...analyzing your plasmids. Virus Protocols for titering and testing your virus preparations. Antibodies ...protocol Fluorescence Titering Assay for Lentivirus Quantify virus using fluorescence measurements Colony...Colony Formation Titering Assay for Lentivirus Quantify virus by counting antibiotic resistant colonies Generating...physical titer of your adeno-associated virus preparation Lentivirus Titration by ddPCR Use ddPCR to measure...consortium for targeting human and mouse genes. Virus Name Description Link to Video General Transfection...Production in HEK293 Cells Produce adeno-associated virus to deliver your gene of to the target of your choice...Gradient Ultracentrifugation Purify adeno-associated virus from a preparation produced in mammalian cells Watch...

...Formation Titering Assay for Lentivirus You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols...the seeding of the cells at the same time as the virus-mediated transduction. For some cell lines, transduction...into 20 mL of media. If using freshly collected virus, filter through a 0.45 μm polyethersulfone filter...freeze-thaw. If you are freezing and aliquoting virus, it is recommended that you titer from the frozen...titer associated with freeze-thaw. If using frozen virus, thaw the lentiviral aliquot on ice prior to use...Complete Containing 10 μg/mL Polybrene (μL) Volume of Virus Added to Plate (μL) Volume of Cells Added to Plate...Plate (μL) Final Viral Dilution 1:10 100 of Stock Virus 900 150 1,350 1:100 1:100 100 of 1:10 900 150 1,...

...cells with fluorescence V V = Virus Volume, mL For example: If 15 µL of virus added to 150,000 cells resulted...Fluorescence Titering Assay for Lentivirus You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols...the cells overnight. If using freshly collected virus, filter through a 0.45 μm polyethersulfone filter...freeze-thaw. If you are freezing and aliquoting virus, it is recommended that you titer from the frozen...titer associated with freeze-thaw. If using frozen virus, rapidly thaw the lentiviral aliquot on ice prior...the dilution factors (method 1) or the volume of virus (method 2): Method 1 Method 2 $$T = {N*F*D\over ...used in downstream applications. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please...

...ultracentrifugation to get pure adeno-associated virus (AAV) preps for use in your research....Ultracentrifugation You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Introduction This protocol...an isomolar density gradient medium suitable for virus purification and isolation of cells, organelles,... remove iodixanol prior to use of your purified virus. Nonetheless, we recommend performing a buffer exchange...fewer fractions once you have a good idea when the virus elutes from the gradients. In a biosafety cabinet..., or you will lose a significant amount of your virus. When the first fraction is collected, move the ... off the walls of the filter to recover as much virus as possible. Store at 4 °C for short term (2 weeks...

...they vastly outnumber the virus particles. Incubate the cells with the virus for 48–72 h. Gently aspirate...Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service Introduction This protocol can be...downstream applications. If you’ve titered your virus beforehand, you can narrow this range according ...6-well plate will hold 5 dilutions plus one 'no virus' control well). Perform a "reverse transduction"...added to the wells that already contain 0.5 mL of virus solutions at various dilution. Make sure to use ...Transducing too many cells relative to the number of virus particles reduces the transduction efficiency, resulting...Typically, cells transduced with lower dilutions of the virus will have higher levels of expression. Consider ...

...replace with fresh media. Day 3-4 (am): Harvest virus Equipment Class II, Type A2 Biological Safety Cabinet...DMEM Complete or OptiPro SFM. Incubate the cells. Virus can be harvested at 48, 72, and 96 h post transfection... Protocols Lentivirus Production Lentivirus Production You may also ...this protocol to generate lentivirus Protocols...Introduction This protocol can be used to produce lentivirus from a lentiviral vector transfected into 293T...lines or transfection reagents. Once produced, lentivirus can be used for a variety of downstream applications...

... C = Copies/µL D = Dilution factor of virus V = Volume of virus in reaction mix (4 µL) Sample Data When...Titration by qPCR AAV Viral Service Viral Vector Guides Virus Blog Posts Molecular Biology Reference Introduction...Water Procedure Preparation Before handling any viruses, get materials ready. Ensure that primers, probe...contamination of the PCR reagents and the NTC with virus. Pre-warm the 96-well plate sealer by gently touching...plate and pipette 5–10 times to mix. Dilute the virus as follows: Mix dilutions thoroughly, but pipette...

...this is likely to shear the envelope of your virus. o. Virus may be stored at 4°C for a few days, but should...decrease the efficiency of the virus, so Addgene recommends that you use the virus immediately or aliquot the...0.05-1 mL virus (add 0.5 mL for a high MOI, and 0.1 mL for a low MOI). Scale the amount of virus added depending....g. BL2+) suitable for handling HIV-derivative viruses. For transient knockdown of protein expression,...protocol can be scaled to produce different amounts of virus as needed. Day 1: a. For each plasmid to be transfected...infection time to between 4 – 20 hours. Remove the virus-containing media and replace with fresh media. Do...DS et. al. Cell . 2003. 115:199-208. PubMed . Lentivirus-delivered stable gene silencing by RNAi in primary...

...Protocol AAV Purification Purify adeno-associated virus from a preparation produced in mammalian cells AAV...measure the physical titer of your adeno-associated virus preparation. AAV Titration by qPCR Using SYBR Green...

...Follow this protocol to produce adeno-associated virus (AAV) in HEK293 cells....also like... AAV Viral Service Viral Vector Guides Virus Blog Posts Molecular Biology Reference Introduction...allow full precipitation. Precipitation of the viruses can proceed overnight at 4 °C if needed. Transfer...cleared lysate to the tube containing the resuspended virus from step 12 above. Add 50 units of benzonase per...

...Technology You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Introduction The AAV... x 10 13 GC/mL and we use an internal reference virus that is 1 x 10 13 GC/mL. We recommend always using...detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences...

...Transfection You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service Introduction...cell line is critical for obtaining high levels of virus. HEK293T cells should be split 3 times a week: Monday...

...antibody-expressing plasmids containing the Epstein-Barr virus origin of replication use a HEK293 line stable expressing...

...Dilution You may also like... Viral Vector Guides Virus Blog Posts Addgene Protocols Viral Service Introduction... pool of stable cells. Transducing cells with lentivirus results in a heterogenous polyclonal population...protocol for generating stable cell lines with lentivirus . Day 0: (optional) Seed cells for the generation...protocol for generating stable cell lines with lentivirus . Pro-Tip Because the polyclonal cell pool is...