...induces mutation at rates much higher than the parental vectors. pC-GoldyTALEN directs expression of TALENs...

...making Integrase deficient lentivirus Rawlings , Scharenberg 12251 pMDLg/pRRE 3rd Packaging Contains Gag and...

...Their ability to self-renew or replicate while maintaining the undifferentiated “stem” state and, Their...to self-renew is greatly diminished. Embryonic and adult stem cells can be used for different research...contributed by Kusumika (Kushi) Mukherjee, editor of Trends in Pharmacological Sciences, a Cell Press reviews...special types of cells that can develop or “differentiate” into more specialized cells with specific functions...function adequately [1]. Stem cells’ ability to differentiate into multiple cell types makes them useful models...Their potency or capability to differentiate into specialized cell types [2]. We general speak of 4 levels... cell has the ability to produce all of the differentiated cells in an organism, including the embryonic...

...comes the need for new conference room names. The Addgenie’s favorite conference room naming scheme? Gene...largest conference room at Addgene is named Sonic Hedgehog but this is not a direct reference to the video...community. Not surprising, this is where most of our conference room names originated from. Let’s take a look...Groucho, a transcriptional co-repressor The first conference room, that is actually used as a break room for...diffuses down a concentration gradient exerting a different effect on the developing embryo depending on its...required for spermatogenesis One of our smaller conference rooms is named Cookie Monster. cookie monster... bagpipe, two genes important for mesoderm differentiation Now, let's turn tinman and bagpipe, two genes...

...made from a selected set of plasmids that are currently in our repository. AAV are distributed as 100 ...

...788679 Lin R, Wang R, Yuan J, Feng Q, Zhou Y, Zeng S, Ren M, Jiang S, Ni H, Zhou C, Gong H, Luo M (2018) Cell-type-specific...in decreased efficiency of recombination and preferentially leads to gene constructs where the stop cassette...likely generalizable to other systems as well. References: del Valle Rodríguez A, Didiano D, Desplan C...plasmids at Addgene Read our Molecular Biology Reference ...

...10.1128/jvi.02012-06 Ou X, Liu Y, Lei X, Li P, Mi D, Ren L, Guo L, Guo R, Chen T, Hu J, Xiang Z, Mu Z, Chen... investigate the susceptibility of cells from different organisms to SARS-CoV-2 (Chu et al., 2020). An... of the scientific research infrastructure. References Chu H, Chan JF-W, Yuen TT-T, Shuai H, Yuan S,...(2006) Is there an ideal animal model for SARS? Trends in Microbiology 14:299–303 . https://doi.org/10.1016...

...10535 (2018). 1 10.1074/jbc.TM118.000372 Yang, H., Ren, S., Yu, S., Pan, H., et al. Methods favoring homology-directed...park. Happy editing!   Resources and References References Yu, Y., Guo, Y., Tian, Qigi, Lan, Y., et...overhangs at the break site – an early HR step (Moreno-Mateos, et al., Nat Com). Another method used ...the break, potentially shuttling repair down a different pathway (Charpentier et al., Nat Com).   ...Cas9 delivery. eLIFE. (2014) 10.7554/eLife.04766 Moreno-Mateos, M. A., Fernandez, J. P., Rouet, R., Vejnar...

...-023-02050-w Yi, Z., Zhang, X., Wei, X., Li, J., Ren, J., Zhang, X., Zhang, Y., Tang, H., Chang, X., Yu...) enzymes. Various other proteins included in different CGBE tools allow the researcher to direct the ...component of the nucleoside inosine. Created with BioRender.com. Inosine excision leads to the first adenine...a translesion synthesis polymerase known to preferentially incorporate A bases across from abasic sites...indicate natural cellular processes. Created with BioRender.com. The first deaminase-free base editor allows... Figure 3: Mechanism of gGBE. Created with BioRender.com. Base excision editing extends to thymine...recognize thymine (T) or cytosine (C). Several different teams succeeded with this approach around the ...

...Educational Resources Molecular Biology Reference Molecular Biology Reference Jump To: Molecular Biology Basics...Addgene's molecular biology reference guide, including information on molecular genetics, plasmids, sequencing...verification Resources Genetic Code Webpage and Blog References Protocols Origins of Molecular Genetics The concept... Joshua Lederberg coined the term plasmid, in reference to any extrachromosomal heritable determinant....cells contain the same genetic information as the parental cell, and are thus termed clones of the original...molecular biologist’s toolbox. To learn more about different types of cloning methods check out our guide on...promoter (e.g., a liver-specific promoter). The strength of the promoter is also important for controlling...

...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...