...Addgene AAV Production protocol, I take a lazier approach for harvesting my AAV. I skip the PEG precipitation...the PEG will separate into two phases. If this happens, the solution can be cooled and the two phases ...immediately before iodixanol purification. This approach gives me a bit of flexibility since I can store...

...The CRISPR corner New ready-to-use viral preps Mapping antibody escape in SARS-CoV-2 by mutational scanning... sequencing. From Starr et al Cell., 2020. (B) Mapping antibody-escape mutations with the yeast display...specific anti-SARS-CoV-2 antibodies (Fig 1B). This approach can augment viral surveillance by enabling rapid...

...are, you’ll even have DNA left over for other applications, like PCR, cloning, transfection, or long-term...wash step or use a different strain. For most applications, Addgene uses and recommends DH5𝝰! This strain...your E. coli to ensure it is suitable for your application. We have previously reviewed many common lab ...

...(2015). “A Scalable Genome-Editing Based Approach for Mapping Multiprotein Complexes in Human Cells.” ...lies in the diversity of nucleases and their applications. It will be interesting to see what new editing...

... map the internal organization of a cell. This mapping can be done by using antibodies targeted to the...meaningful way it is important to choose the appropriate quantification methods and tools. It is usually...Pages 2. Cooper GM. 2000. The Cell: A Molecular Approach. 2nd Edition. 3.  CureFFI.org. Cell Biology 04...

...virtually any genetic locus when complexed with an appropriately designed small RNA, or guide RNA (gRNA). The...expression with truncated gRNAs An alternative approach is to modulate Cas9’s nuclease activity by modifying...engineering parameters via a library-on-library approach." Nature methods 12.9 (2015): 823-826. PubMed ...

...Cascade binding and subsequent DNA degradation appears to be conserved. This DNA degradation portion can... portion of DNA using only a single gRNA. Cas3 appears to have fewer off-target alterations, likely due...Mashimo, T. (2022). Genome editing technology and applications with the type I CRISPR system. Gene and Genome...

...machine learning (PRIDICT, PRIDICT2.0).   How to apply PRIDICT in prime editing experiments Prediction ...pegRNA efficiencies with pridict.it. This approach leverages the strengths of PRIDICT/PRIDICT2.0 ... computational biology. His expertise includes applying machine learning techniques to make sense of biology...

...of proteins. But they are still flat. To truly appreciate the 3D structure of these proteins, it is necessary...molecular viewing software.   In reality, this rarely happens. But if you ever have an opportunity to read one...invariably say something like “Oh,…I didn’t quite appreciate that….” For example, when Tom Steitz was presented...

...reference sequence into the Subject Sequence box that appears. Aligning two nucleotide sequences is probably ...frameshift mutation present in the ORF is readily apparent when viewing blastx results. When using blastx...webservices/#pairwise_sequence_alignment_psa Happy BLASTing! Do you have any tips for using BLAST to...

...PIPs, resulting in a fluorescent readout.   Applications for bimolecular fluorescence complementation...resulting from the assayed PPI. This high-throughput application is much more straightforward to carry out because...without structural information, constructing appropriate controls may requires that you empirically test...

...What the Future Holds and the Scientists Making It Happen, is an exercise in restraint. The very first subheading... primarily on fluorescent proteins and their applications, tackles much broader issues in this brief volume...ago I would have nodded my head knowingly and appreciatively throughout this chapter. Now, these observations...

...the most important is letting people know it’s happening. Aim for 3-4 weeks advance notice for the first...the Addgene bioinformatics journal club! What happens during journal club? There are many common ways....  These variations are suited for different applications and should be carefully chosen for each group...

...Addgene  Choosing an expression system Choosing an appropriate expression vector is the first step in generating...binary gateway vectors and can be used for promoter swapping and generating N and C terminally tagged fusion...phenotypes. For most phenotypic analysis it is appropriate to begin working in the T2 generation. The selection...

... or burn yourself out trying to slog through unappealing work in your precious free time.  And don’t be...- will be required to produce an outcome you’re happy with. Know if you’ll need specialized equipment ...then pick whichever is most accessible or most appealing to you. And if you’re not sure what all the possibilities...

...primers. Find Empty Backbones at Addgene Each approach has advantages and drawbacks, which are summarized...which method you choose, make sure to use the appropriate antibiotic for selection.   Lysing bacteria...to determine their size. Make sure to use an appropriate molecular weight standard for reference and to...

...virulent or antibiotic resistant bacteria, and inappropriate antibiotic usage selects for resistant bacteria...to combat antibiotic resistance using multiple approaches. Major restaurant chains including Chipotle and...bacterial host range. To further potential clinical applications, Ando et al. recently engineered synthetic phages...

..., Alexa Fluor 568, or Alexa Fluor 647 before application to the sample. Staining with this multi-color...Mouse IgG2a TP1129 pTP1005 (3x cysteines) Mouse Kappa TP1170 pTP1174 (1x cysteine) pTP1172 (3x cysteines...labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation. eLife. PubMed...

...which a vaccine is available. However, here it appears that the culprit for these unusual cases was either... the removal of certain adenoviral genes that apparently contribute to virulence (Tollefson et al., 1996...on the Addgene blog Viral Vectors 101: Viral Applications Adenoviral Vector Production and Troubleshooting...

... an ideal way to deliver AAV to the PNS. It’s a happy accident that AAV.PHP.S targets the PNS since it...jumble of fluorescent neurons. A simple but flawed approach to multi-color labeling is to inject a mix of ...morphologic studies), as well as translational applications (mechanistic studies and gene therapy treatments...