...If possible, include an AAV reference sample of known titer. The reference material should have a titer...an internal reference virus that is 1 x 10 13 GC/mL. We recommend always using a reference within 1-log...should observe differences in Ct values that make sense for your dilutions (~3.3 difference Ct for a 10-...This protocol was validated using an internal reference AAV of known titer, 100837-AAV1, and by measuring...master mix should also contain an internal passive reference (typically ROX dye), to normalize non-PCR–related...using this plasmid, but will change if you use a different plasmid. Sample Calculation Size in bp of Addgene...standard dilution. Dilute DNase-treated and AAV reference samples according to the dilution scheme in the...

... DMEM complete + 10 µg/mL polybrene by diluting 20 µL of 10 mg/mL polybrene into 20 mL media. Thaw the...with DMEM complete + 10 µg/mL polybrene, the final concentration of polybrene in each well should be 10 µg...glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene (10 mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 ...21-040-CV (cations can affect the attachment of adherent cells) 0.45 μm polyethersulfone filter, Nalgene... 5mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and FBS lots can promote or inhibit transfection...of the lentivirus in DMEM complete + 10 µg/mL polybrene. Note, this is just a sample of possible dilutions...Lentivirus (μL) Volume of DMEM complete + 10 µg/mL polybrene (µL) 0 0 500 1:5 300 200 1:10 150 350 1:50 30 ...

...μg/mL polybrene. TIP: Polybrene increases the efficiency of viral infection. However, polybrene is toxic.... Hexadimethrine Bromide (Polybrene) Prepare a 1mg/mL solution of polybrene (Sigma-Aldrich catalog #H9268...lentiviral infection and selection G. Safety H. References H.1 Published articles H.2 Web resources I. Appendix...than others. In addition, demonstrating that two different shRNAs that target the same gene can produce the...repeats found in lentiviral plasmids, Addgene currently recommends using a strain that reduces the frequency... plate. The protocol can be scaled to produce different amounts of virus as needed. Day 1: a. For each...In polypropylene microfuge tubes (do NOT use polystyrene tubes), make a cocktail for each transfection...

...complete containing 10 μg/mL polybrene by diluting 20 μL of 10 mg/mL polybrene into 20 mL of media. If using...48–72 h. Pro-Tip Different cells may be more sensitive to lentivirus or to polybrene, in which case this...glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene (10 mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 ...21-040-CV (cations can affect the attachment of adherent cells) 0.1% Crystal Violet, Ward’s Scientific ...5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote or inhibit ... before transduction. Note that in that case, polybrene should only be added at the time of viral transduction...lentivirus into DMEM complete containing 10 μg/mL polybrene: Dilution Volume of Lentivirus or Previous Dilution...

...liquid or separate liquids of different densities is essential to many different experimental procedures, ...likely encounter several different types of centrifuges that can accommodate different sized containers, spin... selecting and using a centrifuge to separate different components in a liquid sample. Protocols...piece of laboratory equipment used to separate different components in a liquid sample. It achieves this...spin at different speeds, or keep samples at specific temperatures. This protocol will cover the general...rotor. How to Centrifuge Now that you know the different parts of a centrifuge, you’re ready to begin using...Conclusion Centrifuges are important tools for many different protocols. Improper use of a centrifuge can damage...

...21-040-CV (cations can affect the attachment of adherent cells) Trypsin EDTA (TrypLE, Thermo Fisher, 12605010...EX0276-1 Benzonase 250 U/µl, Millipore #71205-3 Polybrene 10 mg/mL, Millipore, TR-1003-G Molecular Biology... Pierceable foil heat seal, Bio-Rad, 1814040 Polystyrene reservoirs, VWR, 89094-662 Microcentrifuge tubes...300,000 cells/well in 1350 µL media and 11.1 µg/mL Polybrene into the wells containing the virus and the untransduced...,000 cells in 13.5 mL of media and 11.1 µg/mL Polybrene. Mix the cell suspension well before seeding. ...final volume in the well is 1.5 mL, and the final Polybrene concentration is 10 µg/mL. Mix well and incubate...cartridge. Add 800 µL of droplet generation oil to a polystyrene reagent reservoir. Using the 20–200 µL multichannel...

...Tips Although there are many different types of pipettes that have different mechanisms to disperse liquids...that the same pipette can be used for measuring different samples without cross contamination as long as...performs the same function, the numbers are read differently on each type of pipette. This section will help...Procedure How to Pipette Now that you know the different parts of the pipette, how to select tips, and ...pipette tip. Liquids of various viscosity have different flow rates. The more viscous a liquid, the lower... are pipetting the same amount of liquid into different tubes or into wells of a microplate, you may consider...

...samples have significantly different concentrations, it is possible that the difference between them has affected...Plasmid DNA Background Information During several different stages of molecular cloning, it is important to...particular substance in that liquid. Molecules absorb different wavelengths of light to varying degrees and most... DNA added. 'Blanking' measures the background inherent to the machine and your solvent. If using a NanoDrop...consistent readings from DNA in a buffered solution. Reference Page Top Index...

... Select the Trendline checkbox. Determine the linear equation and the R 2 of the trendline in Google Sheets... The R 2 of the trendline should be between 0.95–1. Use the equation of the trendline to determine the...may need to be adjusted to accommodate slight differences between products. Addgene does not endorse or... Inclusion of this information is solely for transparency intended to support reproducibility in science...where the x-axis is represented. Add a linear trendline for the standards. Google Sheets can calculate...

...Picogreen, or Qubit. Each different DNA measurement type can report slightly different concentrations of DNA...All CRISPR Pooled Libraries Molecular Biology Reference How do I process my Addgene pooled library? Introduction... Pooled libraries contain tens to millions of different plasmids in a single sample. They are often used...only a short region of DNA: guide RNAs targeting different genes in an organism's genome, for example. Amplification...should be avoided as best as possible due to the inherent possibility of altering the composition of the...the library. Bottlenecks, fitness differences, and plasmid recombination can all impact the representation...

...because different components of the specimen will refract and reflect the light. These differences create... of biological research and are found in many different types of laboratories. These tools allow you to...sizes - from phone-sized, foldable microscopes that aren't very powerful (but are cheap and accessible) to... light microscope with labels. Created with BioRender.com. Base Light Source Condenser and Diaphragm Stage...objective performs essentially the same task but has different magnifying powers. When you look at the side of...

...may need to be adjusted to accommodate slight differences between products. Addgene does not endorse or... Inclusion of this information is solely for transparency intended to support reproducibility in science...temperature or 37 °C. The protocol notes when there are different incubation options available. Caution Use the ...76322-134 Pipettes, 10 mL, VWR 89130-898 1 L polystyrene bottle, Corning 430518 PBS, 1X pH 7.4, VWR 45000...and vortex. Pro-Tip You may need to try a few different concentrations of antigen to determine the ideal...into the first three wells of each row, using a different standard solution for each row of a 96-well ELISA...

...glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene (10mg/mL), Millipore TR-1003-G 1X PBS pH 7.4 without...21-040-CV (cations can affect the attachment of adherent cells) 0.45 μm polyethersulfone filter, Nalgene... mL of 100X glutaGRO. Store at 4 °C. Pro-Tips Different brands and lots of FBS can promote or inhibit ...lentivirus into DMEM complete containing 10 μg/mL polybrene. Note, this protocol was developed using low titer...Volume of DMEM complete (μL) Volume of 10mg/mL polybrene (μL) 1:10 150 1348.5 1.5 1:25 60 1438.5 1.5 1:...

...days. Note that growth rate differences can be inferred by the differences in colony size. Figure 2: Cas9...21-040-CV (cations can affect the attachment of adherent cells) Trypsin EDTA (TrypLE, Thermo Fisher 12605...increased serum concentrations, may be optimal for different cell types. Before starting this procedure, be...expression. This protocol has been optimized for adherent cells but can be modified for suspension cells...

...Vortex Reagents Adherent HEK293T cells (ideally AAV-293T clones) Pro-Tip While adherent, these cells are...Vector Guides Virus Blog Posts Molecular Biology Reference Introduction This protocol can be used to produce...1272 cm 2 Heat-inactivated FBS (HI-FBS) Pro-Tip Different brands and lots of FBS can promote or inhibit ...21-040-CV (cations can affect the attachment of adherent cells) 1 mg/mL Polyethylenimine (PEI) 25 kDa MW...

...backbone (See Addgene's Sequencing Primers for reference). Many sequencing cores have a list of common ...recommends using Addgene's sequencing results as a reference for primer design. A good sequencing reaction ...reaction with high background: If you obtain a different sequence than expected and wish to contact Addgene... should I do? Check your trace file first; the apparent mismatch/mutation may be the result of a mis-called...

...accurately sized on a gel and also such that they are different enough in size to be easily resolved from each...approach, it is often a good idea to choose two different enzymes that will give you unique but distinct...In the example below we want to know how to differentiate between two clones that differ only in the ...the expected products from each orientation are different sizes. Simply run the digest products on a gel...

...two different enzymes for adding an insert into a vector (one enzyme on the 5' end and a different enzyme...controls. See Tips and FAQ below for details. Try different vector to insert ratios to optimize the ligation...will tell you exactly how much of each to use. Reference Page Top Index...

...isothermal reaction. With the activities of three different enzymes, the product of a Gibson Assembly is a...mix (isothermal reaction mix) consists of three different enzymes within a single buffer mixture and an ...primers to include in the Gibson reaction are different than with normal PCR primers (Gibson et al. , ... than five fragments at a time. Resources and References New England Biolabs (NEB) (Link opens in a new...

...use one or the other, but there is very little difference between the two. Note: Make sure to use the same... buffer as the one in the gel box (do not mix different buffers and do not use water). Microwave for 1...be in the top portion and your bands will be differentially intense. If this happens, you can just soak...to load 1.0 μg in 10 μL, make 1.1 μg in 11 μL. Reference Page Top Index...