... so if you are interested in learning more about any one of them, please click on the links throughout...with a special emphasis on these five categories. Please note that many of the following cloning methodologies...ends. These fragments are then combined with an exonuclease, a polymerase, and a DNA ligase to carry out ...FastCloning, first described by Chaokun Li and colleagues. Here you simply PCR-amplify your vector and ...When mixed, topoisomerase I provides both the endonuclease and ligase function while 5’ adenosine overhangs...additional enzymes required. FastCloning may be the least expensive of all the options discussed as it only..., as one tube of enzyme should be enough for at least 50 cloning reactions. The ligase cost is comparable...

...University of Zurich), especially with my deep learning-wizard colleague Ahmed Allam. Continued training led to...desired edit, it simply becomes impossible (or at least unreasonable) to test them all. This is where PRIDICT...efficiency and building prediction tools with machine learning (PRIDICT, PRIDICT2.0).   How to apply PRIDICT...prediction tool (ePRIDICT). In summary, machine learning-based prime editing prediction models (such as...biology. His expertise includes applying machine learning techniques to make sense of biology, with a special...SpCas9 activity prediction by DeepSpCas9, a deep learning–based model with high generalization performance... editing efficiency and product purity by deep learning. Nature Biotechnology, 41, 1151–1159. https://...

...the IRES element, scientists have adapted "self-cleaving" 2A peptides into their muliticistronic vectors...mulitple genes from the same mRNA. The term "self-cleaving" is not entirely accurate, as these peptides are...peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence...sequence and the next peptide downstream.4 The "cleavage" occurs between the Glycine and Proline residues found... common 2A peptides employed by scientists. 2A cleavage is universal in eukaryotic cells, and, although...although some scientists report close to 100% cleavage with some of these peptides, no consensus has been reached... added to the 5' end of the peptide to improve cleavage efficiency. How do I get started? If you are looking...

...bioinformatics and HTS, we all had much to learn. And, many wanted to learn more, even those not directly involved...continue such discussions. Beyond the excitement of learning about new cutting edge science, researchers of...in journal clubs. They also have likely had the pleasure of attending both productive and not so productive... (HTS) data we generate and maintain. It became clear that while several people across teams had various...same characteristics as a successful meeting: a clear purpose and effective time management, for starters...feedback you receive include the sense that people learned something new? Did the meeting begin and end on... published science. They also serve as a way to learn about work outside of one's own field in a structured...

...based on the experiments of Kim and colleagues using zinc finger nucleases to harness non-homologous end joining... (DSB) approach at a variety of loci. We were pleasantly surprised by the efficiency of the method. One...polymorphism analysis, allele-specific PCR, T7EN1 cleavage assay, sequencing, RT-qPCR, or immunoblotting ...genetic configurations. Watch the following video to learn Daniel and Matthew's procedures. The video details...genetics of blood cell development and disease. Learn more at his webpage, bauerlab.org.     Matthew Canver...chromosomal deletions in human cells using zinc finger nucleases. Genome Res. 2010 Jan;20(1):81-89. PubMed. 2. ... interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. J.Biol.Chem. 2014 Aug...

...with other proteins or nucleic acids. Read on to learn more about this versatile technique.  Immunoprecipitation... only to the Fc portion of the immunoglobulin, leaving the antigen binding sites free to capture the target...conformations and interactions. For difficult to release proteins, including those in the nucleus, more ... an IP, as proteins in an IP are not denatured (learn more about antibodies here!) When using Protein ... immobilize the antibody, it is recommended to preclear the sample to remove any proteins that may interact...nonspecifically with the capture antibody or Protein A/G. To preclear, incubate the sample as you would in an IP using...interactions from residual non-specific proteins but leaves the target protein/capture antibody intact. The...

...applications. Tag cleavage must be performed after primary purification and requires a second clean up step to...protease cleavage at sites immediately following the tag. Unlike MBP, the GST tag can be cleaved off from...need for an extra clean up step of the tag – a handy feature. If you decide not to cleave the GST tag after...Waugh, 2002). The tag can also be proteolytically cleaved after purification, so as not to perturb protein...to remove the cleaved tag from solution. MBP antibodies are available for detection and/or purification...

...genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33:187–197 . https...we’ll just be covering DNA base editors here. To learn more about RNA base editors head over to this blog...2017). Gam binds the free ends of DSBs, which may lead to cell death rather than NHEJ repair, thus removing...they are expressed well. The Liu lab modified the nuclear localization signals and codon usage BE4 to create...also made an adenine base editor with improved nuclear localization and expression. This base editor was...et al., 2018 BE4max, ArcBe4max, ABEmax Improved nuclear localization and expression Gaudelli et al., ...editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464–471 . https://doi.org/10.1038/...

...CRISPR Featured Topic Page Learn about Non-Homologous End Joining Learn about Base Editing Read 3...can be caused by intracellular factors such as nucleases and reactive oxygen species, or external forces...: The 5’ DNA end of the break is resected by nucleases to create a ssDNA 3’ overhang. This will serve... a primer for DNA repair synthesis, ultimately leading to the formation of double Holliday junctions (...second DSB end. The individual HJs are resolved via cleavage in one of two ways - each junction resolution ...single HJ forms during this process and resolves by cleavage of the crossed strand. While this pathway may ...point to note is that Cas enzymes can continue to cleave DNA if the gRNA/PAM site remains intact. In other...

...different promoters followed by leader sequences (encoding small leader peptides) downstream of which you... been used to date . If you have any questions, please write to us in the comments section.  FX cloning...gene of interest. Translation of the standardized leader peptide prevents secondary structures in the mRNA...Shine-Delgarno sequence found at the 3’ end of the leader sequence. Using a variety of GFP and RFP fusions...Katarzyna, et al. "Small changes in enzyme function can lead to surprisingly large fitness effects during adaptive...reported in the literature include RNA detection, RNA cleavage, and translational repression or activation: ...Choudhury, Rajarshi, et al. "Engineering RNA endonucleases with customized sequence specificities." Nature...

...ideas in the easiest way possible? Are the shots clear enough for the audience? As Addgene’s Science Video...SynBio, it was time to start our Design-Build-Test-Learn cycles. If you’re not familiar with this, it’s a...process!   Figure 1: The Design-Build-Test-Learn cycle adapted for video making.   Cycle One:... pulled it off! Check out the PCR Primer video! Learn: Producing videos like this would prove to be difficult...phones. Test: I loved the end result, but it was clear this wasn’t going to work for our other videos. ...1280x720 resolution at best. Some came in even lower.  Learn: While this works well for social media videos, ...chat with me remotely via an audio chat (Fig. 4).  Learn: This works great for on-camera presentations and...

...classroom or a defined learning program. Learners cannot (theoretically, at least) fully and freely engage... fall under informal learning; however, many others partner with formal learning to engage students at...education that is provided, typically ad hoc, for learners who can choose to engage or disengage with a very..., and their expectations of you, consistent and clear.   And now that we’re all on the same page, we’re...re ready to get into the more exciting parts - learning more about science communication itself! See you...

...This decrease in pH quenches SEP but not DsRed, leading to emission of only red fluorescence. The ratio...ratio of red to green fluorescence allows for clear distinction between vacuolar/lysosomal and cytoplasmic...lysosomes. Therefore, the formation of autophagosomes leads to an increase of yellow puncta (GFF positive/RFP...autophagosome with a lysosome. An increase in autophagy leads to more yellow and red puncta, while a block at ...at fusion of autophagosomes with lysosomes leads to an increase in yellow puncta but a decrease in red ...Addgene! GFP-LC3-RFP-LC3∆G: A fluorescent probe that releases an internal control Another autophagy biosensor... following translation, this fusion protein is cleaved to generate one molecule of GFP-tagged LC3 for ...

...self, too — when talking to friends or dates or colleagues we tell tales about parts of our lives that tend...structure: beginning, end; cause, effect; lesson, learning. Storytelling is a crucial part of interpersonal... telling a good story is something that can be learned, and will serve you well in your career. Story ...you. Make sure to highlight what lessons you’ve learned and what knowledge you’ve gained. Show that as ...approachable. Engage with your audience and try to learn from them. Science is a collective endeavour. Your... your lowest points and highest points and make clear what was difficult and what was easy when appropriate...people enjoy and engage with and what parts you can leave out. Start viewing your work in the form of a story...

... said Claudia OuYang, BeHEARD Co-Director in a release. The research teams studying four of those rare...disease caused by mutations in the OCRL1 gene that leads to the early death of young patients as their kidneys...processes involving cell membrane dynamics. When they learned there was a disease associated with the gene, it...Pigmentosum Hendrik De Buhr (top right) and his colleagues at the Wistar Institute in Philadelphia, PA will...pigmentosum (XP). This recessive genetic condition leaves patients incredibly sensitive to sunlight and prone... help them to understand how different mutations lead to differences in the symptoms experienced by AHC...AHC patients. Waardenburg Syndrome Last but not least, Meng-Shin Shiao (right) of Mahidol University’s...

...the four strands of the cleaved duplex, while the fourth strand (the released strand in Figure 1 below...Corn’s lab. CRISPR-Cas9 (Cas9) is an RNA-guided nuclease that targets and cuts genomic DNA. The interplay...discovered that the catalytically active form of the nuclease dissociates very slowly after introducing a cut...experiments with more detailed investigations into the release of DNA post-cut. Using substrate DNA labeled on...single stranded donor DNA: Complementary to the released strand, 127bp in length, With 36bp on the PAM-...disastrous (i.e. if breaking the gene is worse than leaving the mutation alone). CRISPR delivery by nucleofection...on the Addgene Blog Brush Up on Your HDR Basics Learn How to Design Your gRNA Find the Ideal CRISPR Software...

...functions both as a restriction endonuclease and as a ligase by cleaving and rejoining supercoiled DNA ...for the amplification step since Taq polymerase leaves a single deoxyadenosine (A) at the 3' ends of PCR...breakage is stored as a covalent bond between the cleaved 3′ DNA strand and a tyrosyl residue of topoisomerase...covalent bond thus joining the two DNA strands and releasing topoisomerase (2).  These days commercially available...PCR product. If you need to decrease error rate, please use one of these methods to ensure your insert ...extra salt to your reaction: Topoisomerase I is released from the vector when the PCR product and vector... Generate Insertion and Deletions in a Plasmids Learn How to Verify Your Plasmid Resources on Addgene.org...

...applications. We recently released a new variant of jGCaMP7 (keep reading) and will be releasing more variants in...viral vectors. Here are some of the AAV we have released so far in 2019. You can also browse our entire...pAAV-mDlx-NLS-mRuby2 CAG-NLS-GFP pAAV-hSyn-mCherry Read on to learn more!  Calcium sensor AAV The GFP-based calcium...inhibitors that respond to blue light. We recently released a constitutive (pAAV-Syn-Archon1-KGC-GFP-ER2) ...coming soon! These vectors listed below should be released at Addgene in the next 6 months, pending quality... particular catalog item is available for order please email [email protected] with the vector name and...viral vectors 101 eBook Resources on Addgene.org Learn about Addgene's viral service Find viral protocols...

...and knew it was time to leave. I really enjoyed my time in the lab, my colleagues and what I was researching...way to learn something about marketing would be to start reading a broad simple overview, learn some jargon...www.phdadvice.com. I sold out, well in part that’s what leaving academia feels like for a lot of researchers. For...years I struggled with the questions of “Will I leave academia?” and "What type of science careers would...Career Achievements”, and “Academic Achievements”. Leaning my CV more towards me as a product and not me as...important. For my Postdoc position, I tried to read at least 50 papers on the topic before I went for the interview...

...switch that expresses nuclear mCherry in the absence of Cre, and expresses nuclear EGFP in the presence...viral vectors. Here are some of the AAV we have released in the last few months. You can also browse our...testing purposes. To address this, we recently released our serotype testing AAV, which are small (20 ...continue to add more controls, and have recently released these controls: pAAV-hSyn-DIO-EGFP (50457-AAV5...and recombinase-dependent vectors. We recently released several recombinases in various serotypes, and...grow this collection. Of note, we are excited to release a Flp-dependent Cre AAV in AAV9 and AAVrg. See ...viral vectors 101 eBook Resources on Addgene.org Learn about Addgene's viral service Find viral protocols...