...vectors are then packaged into AAV virions via transient transfection of 293T cells with helpers, and used...disadvantages to the AAV-based approach that must be considered when deciding whether to use AAV or CRISPR for... is a Professor of Oncology at the Lombardi Comprehensive Cancer Center, Georgetown School of Medicine...

...States government? Politics and science are intrinsically linked: governments exist in reality and science...include science in decision making, it will be considered a success. Below are resources for the march ... “politicized science” ABC News: White House considering gutting 38 EPA programs: draft budget  Science...

...donor (e.g., sister chromatid, plasmid, etc.), and consists of three pathways: classical double-strand break...you need to generate a knock-in.   General considerations for designing a repair template to create mutations...cells than their plasmid counterparts. Another consideration for dsDNA templates is their ability to randomly...

... social science to an audience that I thought consisted of all biologists. The social scientists in the...a solution or overcame obstacles. Take time to consider the order of your narrative. Scientists do this...are worried that this might sound condescending, consider explaining the term on a slide but not reading...

...repetitive sequences, but if present, a minor extension can usually ensure that the 3’-base(s) do not ...manufacturers’ PCR protocol. Be sure to adjust the extension time to match the size of your template. Amplification...PubMed PMID: 2448875. 4. Xia, Yongzhen, et al. "New insights into the QuikChangeTM process guide the use of...

...really there are no positive results to be found. Consider the hypothetical situation in Figure 1 where many... (or pre-publish) sound data even if it isn’t considered immediately impactful. Down the line we may see...Scientific Figures into Dashboards and Share Your Insights Who gives a tweet? 9 facts about scientists on...

...industry positions. There were, however, some downsides to working in a large commercial company. In my...sometimes go unnoticed at large organizations. Another downside to large commercial companies which are reliant... treatment outcomes. I would say that the only downside of modern genetics from the perspective of somebody...

... the discovery of microbial rhodopsins, light-sensitive proteins that absorb photons for energy conversion... neurons in the brain are not naturally light-sensitive, so the selective expression of opsin genes in...neuronal subpopulations, and this represents a major downside in optogenetics (Belzung et al., 2014). This could...

...professional training after earning a doctorate Considered a "stepping stone" position to become a professor... lab Undergraduate student Roles and responsibilities can vary "Lab assistants" usually wash glassware...1-2 years) Often a gap-year position taken to transition to the next career stage (ex: grad school, med...

...steps 2-4 25-30 times. Final Extension for 5 minutes at 72°C: A final extension to fill-in any protruding...below the primer melting temperature (Tm). Set extension step at 1-2 minutes per kilobase of product depending... instructions for specific instructions about extension time and temperatures. Initial Denaturation for...°C. Repeat steps 8-10 for 25-30 cycles. Final Extension for 5 minutes at 72°C. Run 2 μL on a gel to check...The rate of DNA synthesis is ~1-2 kb/min. The extension time can be adjusted according to the length of... temperature around 70°C and is the molecule responsible for driving the DNA synthesis. Sterile dH 2 O...

...the surface or the inside of your cells. While proteins themselves can emit intrinsic fluorescence when...fixable dye stains that react with free amines on and inside the cells and are thus typically used for distinguishing...

...sequences can be optimized for specificity and sensitivity, for example by changing certain amino acids ... that can pass through each well - the optical density (OD) - of the known and experimental wells is measured...is used to detect proteins on the surface of or inside whole cells. Antibodies - typically, primary antibodies...

...developed in David Liu’s lab in 2019, has added considerably more precision and flexibility to the CRISPR...engages the prime editor protein. The prime editor consists of a Cas9 nickase fused to a reverse transcriptase...mismatch repair   ✓   ✓ ~ ~ ~ Transiently co-express mismatch repair inhibitor MLH1dn ...

...people will have different roles, titles, and responsibilities during their careers - being aware of this...a prestigious institution: This criterion is consistently under scrutiny. USCIS wants you to prove that...review at the request of your supervisor is not considered peer review. The peer review request must be ...

...culture of bacteria transformed with your plasmid Resuspension buffer Denaturing solution Renaturing solution... This step gets all of the bacteria back into suspension, but within a smaller volume of buffer that is...Prepare the following solutions: Solution I - Resuspension Buffer 25 mM Tris-HCl (pH 8) 50 mM glucose 10...phase Middle phase - A white layer may appear, consisting of precipitated protein particles Bottom phase... mM Tris-HCl pH 8, 0.1 mM EDTA). Pro-Tip DNA resuspension can take time, it is a good idea to let it sit...eliminate RNA contamination. This is included in the resuspension buffer of most kits. Reference Page Top Index...

...monoclonal line could be generated at any time. Considerations Before You Start Some cell lines tend not to...optimized for adherent cells but can be modified for suspension cells. The number of monoclonal lines generated... this, you are seeding the plate at an average density of 0.5 cells/well. Seeding an average of 0.5 cells...Figure 1: Generation of monoclonal cell lines from expansion of individual A549 cells stably expressing Cas9... in individual wells of a 96-well plate after expansion of single cells for 13 days. Note that growth ...stable cell pool (pool) and from individual cell expansions (monoclonal, 1-5). Wild-type A549 cells do not...

...liquid culture is capable of supporting a higher density of bacteria and is used to grow up sufficient numbers...need to grow for a longer time to get the correct density of bacteria since they will grow more slowly at...specific protocol and conduct an OD600 to measure the density of your culture if needed. A good negative control...culture. More aeration may help to increase the density of the culture. Normally cultures shake at 150 ... this to 350 - 400 rpm to obtain a higher cell density. Reference Page Top Index...

...applications. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that you are... in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health of the target cell... pipetting or inverting. Aliquot 2 mL of cell suspension into each well of the 6-well dish. Incubate the...anticipate that your viral stock will be higher titer, consider additional dilutions. Dilution Volume of Lentivirus...cells in each well. When calculating titer, only consider wells with less than 40% fluorescent cells. Titering...

...gels. They are shared through Addgene without licensing restrictions. Use these instructions to prepare... genome, many applications in biotech, and is considered the most studied Gram-positive bacterium, many...University of Minnesota recently deposited a comprehensive set of plasmids that can be used for either ...

...you plan PEI transfections for AAV packaging, considering using this PEI Calculator. AAV packaging typically...Additional Resources on the Addgene Blog Important Considerations When Using AAVs AAVs Are Great Tools for Gene...