... Addgenie Julian Taylor-Parker explains why you might want to use a specialized E. coli strain for protein... to an entirely new career path, a career coach might be able to help you. Head Addgenie Joanne Kamens...genome, you need to check whether you’ve made the right one. In this post, Addgenie Melina Fan guides us...guides us through some common controls and why you might need them. Happy Reading!   Resources on the Addgene...

...This guest blog post was contributed by Seán Mac Fhearraigh, PhD, of www.phdadvice.com. I sold out, well ...Like a considerable number of researchers, I had high hopes of securing tenure in a British or Irish University...interviewed for 1 hour so having a broad enough insight was going to help me push both my scientific and...death assays. You can find Seán on twitter @Macfhearraigh.    Read more posts about Scientific Career ...

...amplification and verification processes we use to ensure high-quality library distribution. You can also use these...amplification 101 Library amplification seems straightforward: E. coli bacteria are transformed with the ...a typical library, Addgene usually does four to eight electroporation reactions using a total of ~400ng.... Analyzing the reads is, again, seemingly straightforward – you need to count how many times each of ...

...to 3-loci MPE with similar high efficiencies, which is scalable for a higher number of MPE. Therapeutic...leader sequences of a given tRNA to achieve the highest efficiencies of multiplex editing at each site....Cas9-dependent off-target editing and do not cause higher Cas9-independent off-target editing in comparison...

...involves measuring the absorbance/transmission of light through a liquid to determine the concentration ...liquid. Molecules absorb different wavelengths of light to different degrees and many molecules have a specific...method takes more time to set up in the lab, you might not have to do the calculation yourself as many ...disadvantages of each method and when one method might be more suited than another. Additional resources...

...sequences are from AAV2 because they demonstrate high promoter activity (Earley et al. 2020), but non-...downstream AAV applications.  Tips & FAQs Due to their high GC content and hairpin structures, ITRs are prone...rapidly replicate in a growth culture and result in a high percentage of plasmid DNA with mutated ITR sequences...Next-generation sequencing and Sanger sequencing might fail to correctly read through ITR sequences. GC...

...is not just a question of being honest and straightforward, it’s an important part of being a “good scientific...work to figure them out. But, when they come to light, it’s clear that what the researchers thought was...still missing is a clear alignment of incentives. Right now, researchers are not strongly rewarded for being.... But it ought to be possible, and if we get it right, everyone in the scientific enterprise will benefit...

...protocol, get a result and hope it’s close to the right answer because if it’s not, well, your grade is ...the data look like... when it looks good, that’s a high that can last for several days.” Working with CRISPR...Doench thinks that the future of CRISPR will be in high throughput functional characterization of specific...plasmids, figure out what they do and what they might be able to do and to organize it and catalogue it...

...have purified plasmid DNA, this method can be done right in your lab in less than a day. Diagnostic restriction...sequence if necessary. The example plasmid on the right has a total size of 7.3kb, including a 1.2 kb insert...recognition sequence. Most enzyme manufacturers make High Fidelity versions of the endonucleases and/or supply...you to visualize the DNA under ultraviolet (UV) light. Don’t forget to add loading buffer to your digest...

...instrumental in helping us establish consistent, high-quality protocols for lentiviral production. Bryan...Janelia Research Campus have provided valuable insight into AAV production. Finally, we are blessed to...surveyed hundreds of scientists about various ways we might expand Addgene’s services. There was an overwhelmingly...helps scientists and allows us to maintain Addgene’s high quality standards?” Under the leadership of Addgene...

...succeed in communicating. Even the most rigorous, insightful, and novel scientific research will be wasted...your past work has prepared you to contribute at a high level to their team.  When you're writing a grant...anyone else in the world to do it.  In the lab, it might mean persuasively articulating why the results you've...to be reviewed by interdisciplinary panels.  And highly successful scientists need to communicate with ...

...labeling tubes never takes longer than 5 minutes, right?) As a result, I’d always end up stressed and rushing...add in personalized steps like “check drawers for light-sensitive reagents” or “re-sterilize now.” Have ...Moving your reagents from your left side to your right as you use them can help you remember what's been... Fig. 2: Blugene moves reagents from left to right as they use them.    Prevent Interruptions If...

...Viral integration of a construct is also reliably higher than traditional transfection and random genomic...transfection method. This is because viral systems have a high infection frequency and the viral titer (the number...responses in addition to antibody response 2) very high immunogenicity 3) and long-lasting immune response...treatment and gene therapy have also been in the limelight. The FDA approved the first oncolytic viral therapy...

...BioBuilder Educational Foundation in partnership with high school teachers to boost interest and understanding...what started in Cambridge with a handful of local high school teachers receiving training modules in synthetic...fall:  The BioBuilderClub BioBuilderClub engages high school teams around the world who combine engineering...and STEM-loving students include The BioBuilder High School Apprenticeship, preparing local (Massachusetts...

...disease. CRISPR has made genomic SDM relatively straightforward, but plasmid-based SDM has lagged behind. While...well, but poor template digestion can result in a high number of background colonies carrying the unmodified...plasmid is not maintained. Recombination efficiency is high enough to negate the need for selection usually ...introduce mutations. Using commercially available high-fidelity polymerases such as KOD and Phusion, which...

...Mendenhall: The benefits are two-fold: CRISPR/Cas9 offers high efficiency of inducing double strand breaks and ...weeks. CRISPR saves you a couple of months. It’s higher throughput and faster. It’s also easy to run things...transcription factors, but there are many reasons you might want a system like this. It’s actually surprising...with this antibody problem. We thought it was the right thing to do to put these out there. It’s a simple...

...interested in following? Someone you admire for their insight and way of expressing it in meetings? The more ...advice. Your family and friends know you well and might have good ideas for who you should connect with....cautious and make sure to choose one that comes highly recommended from someone you trust. A good coach...compliment to ask someone to share their wisdom and they might just not have the time. Thank them anyway and move...

...neutral pH compartment, ­­like the cytosol, to the higher pH of the vacuole. Read on to learn more about ...enzyme. Pho8 phosphatase activity or the molecular weight shift of Pho8 from its uncleaved to cleaved form... dual color-emission biosensor named after the brightly-coloured Australian parrot. It’s comprised of ...Oligomerization Seeing Red: Simple GFP Photoconversion Light Sheet Fluorescence Microscopy Additional Resources...

...cyan, or magenta light. A gRNA with all 3 distinct hairpin sequences produces white light. Created with ...from red, blue, and green channels overlaid on a bright-field image. Image from Ma et al, 2016. Using... A gRNA with all three hairpins generates white light. All 6 gRNAs can be expressed in a single vector...

...concentrations. Higher IPTG concentration leads to higher RepA expression which results in high plasmid copy...repressor because TALEs can be programmed to bind tightly to arbitrary DNA sequences and have been shown ...