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...multiplexed using tRNAs, within the lentiviral 3’ long terminal repeats (LTR). The 3’ LTR is duplicated during...the lentiviral insert was minimized to reduce recombination and avoid separation of the multiplexed elements...fold improvement in detection efficiency over the original CROPseq. Find CROPseq-multi plasmids here Walton...developed a new prime editor that incorporates La’s N-terminal domain, PE7 (Figure 2).  PE7 shows enhanced editing...-2 research   By Ashley Waldron Ready-to-use recombinant antibodies were recently added to the Addgene...Image from Addgene. These antibodies are recombinant versions of mouse monoclonal antibodies generated...2021). Check out their paper for details on the original antibodies. Find Anti-SARS-CoV-2 Antibodies here...

...homologous recombination efficiency to reduce the number of plants you need to grow to get a recombinant because...solicitation requires funded labs to rapidly disseminate protocols, methods, data, and tools that result...published, but to engage in ongoing, real-time dissemination of information and materials. Many groups publicized...labs all include materials sharing in their dissemination plans and in particular, that they all share...organismal systems described at this meeting were extraordinarily diverse. Plans to develop tools to study their...   Genus Microtus (Zoe Donaldson) Voles have fascinating social behavior. For example, two similar species...because they have only 1,700 genes total). Hydra (Celina Juliano) This is a known stem cell and regeneration...

...AAV particle. Finally, the precision of the assay can vary up to two fold for the final titer value, which... of 1:1 (Zeltner et al., 2010). However, for recombinant AAV2, the same study reported a physical-to-infectious...2010), while another reported that only 20% of recombinant AAV2 particles were full, compared to 50% of ...annealing can be affected by the presence of protein contaminants, which is a factor in AAV titration since the... be as important as the relative titer values. Finally, because physical titer does not give an indication...packaging of genomes can limit production of recombinant AAV-2." Gene therapy 6.7 (1999). PubMed PMID:... Lock, Martin, et al. "Characterization of a recombinant adeno-associated virus type 2 Reference Standard...

... at Mount Sinai Hospital, Toronto, Canada where he studies protein-serine/threonine kinases implicated...detect possible errors in assumptions, use of inappropriate statistical methods, omission of some data, ...tools and assumptions to reveal findings that the originator was oblivious to, no matter how much time they...productivity”, so this is unlikely. Usually, the originating scientist and colleagues are privileged in viewing...generators. This practice doesn’t appear to harm the originating researchers, even if they are occasionally beaten...data are likely, at least in part, due to our inadequate cataloguing of how results were obtained and ...multiple, complex panels. PLOS, itself, has not finalized its definition of necessary data requirements ...

...possibility of carrying over contaminants from the plasmid prep process into the final plasmid eluate. Possible...designed with environmental sustainability in mind (Table 2). Table 2: Sustainability features highlighted in...more importantly, the DNA needs to be free of contaminants, such as bacterial endotoxins, that could affect...Possible contaminants can sneak in from almost any step of the process and include things like genomic DNA...by excess material, leading to less DNA in your final eluate. Bead-based kits can be more flexible in ...impact, check out Addgene’s blog post on bringing sustainable practices for the lab. Plastic consumables (including...by more than half 70% recycled materials Sustainable forestry certification Starch-based compostable...

... its destination plasmids. In the destination plasmid, the ccdB gene is flanked by recombination sites...introduce a gene of interest into the destination plasmid, recombination sites must first be added to flank...compounds needed for growth, or auxotrophy, the inability to synthesize a compound needed to grow. Auxotrophy...usually something toxic. This gene is found on the original plasmid and either the insertion of a DNA fragment...flank the gene. Successful recombination and integration of the gene of interest at these sites in the plasmid...

...Minimizing off-target editing ABEs were originally developed from RNA deaminases, using directed evolution to get...DNA-targeting technology with a fused cytidine deaminase to create C→T base edits at a target locus. A ...characterized this RNA-editing activity and developed a deaminase variant with a V106W mutation, which reduced RNA...directed evolution, introducing mutations to the deaminase subunit to produce a series of ABE8s that were...editing. Base editing in the present—and future The final base editor that was administered to KJ was designated...J., Liquori, A. J., Murray, R., Packer, M. S., Rinaldi, C., Slaymaker, I. M., Yen, J., Young, L. E., &...sciadv.aax5717  Richter, M. F., Zhao, K. T., Eton, E., Lapinaite, A., Newby, G. A., Thuronyi, B. W., Wilson, C....

...and practicing my SciComm skills and I had to coordinate with other people for a variety of projects. ...conceptual chunks: prioritization, planning, and procrastination. Time management through prioritiziation Every... more time for that task the next time around. Finally, when prioritizing, you should always be aware ...Susanna’s post Upload Eric’s post and give it a final read Browse list of previous depositors from Boston...Eric before doing anything else. Preventing procrastination Even if you’ve done all the prioritizing and...and planning in the world, procrastination can easily prevent you from making progress on a task. For ...few things I’ve found useful to avoid such procrastination: Remove distractions: Probably the easiest...

...colonies, and screened for the desired modification. Finally, the positive clones are sequenced to confirm the...plasmid. Although it is commonly believed that the final products of the PCR reaction are pieces of double...recent data (see Xia et al., 2015) suggests that the final PCR products are pieces of linear, double stranded...These homologous ends recombine in vivo to give the final circular plasmid product pictured here. B) Screening... of DMSO to the PCR reaction (usually around 3% final concentration). The DMSO reduces secondary structures...using a methylation-dependent enzyme (DpnI) to eliminate the parent plasmid from the PCR products, the ...duplications (slightly larger bands sizes relative to the original template). Due to the small difference in size...

... EGFP Recombinase AAV Finally, we are excited to add Flp-expression AAV to our recombinase AAV inventory...inventory. Flp is another useful recombinase that can be used in combination with Cre for intersectional control...Flp-expressing AAV. See our recombinase AAV inventory here New recombinases Plasmid Serotype Name ... µL size serotype testing) pAAV-hSyn-EGFP Recombinases Plasmid Serotype Name 55634  AAV1  pAAV-EF1a-mCherry-IRES-Flpo...

...yeast strains S. cerevisiae and S. pombe have dominated the research scene. But what about the other yeasts...promoter, AOX1, was co-opted for the expression of recombinant proteins by the pharmaceutical and biotech industries...efficient at protein secretion, making harvesting recombinant proteins a cinch. This is because it doesn’t ...secrete its own endogenous proteins, making the recombinant protein its major secretion. Beyond sheer protein... good for post-translational modification of recombinant protein products, specifically glycosylation....glycosylation machinery of K. phaffii lacks certain terminal mannose glycopeptides (which most other yeast ...phaffii, relative to S. cerevisiae, is that it predominantly uses non-homologous end joining (NHEJ) to perform...

... TadA-7.10 version with a faster deamination rate so that deamination can happen before Cas homologs let...fused to a DNA-modifying enzyme, in this case a deaminase. Base changes from C•G-to-T•A are mediated by ... modifying enzyme to the target site, and the deaminase induces the DNA base change without a DNA double-strand... is ABE7.10 which contains the deoxyadenosine deaminase, TadA-7.10 (Gaudelli et al., 2017). The idea was...stop codons within T7 RNAP. A phage encoding the deaminase TadA-7.10 A mutagenesis plasmid controlling the...much higher base editing activity compared to the original TadA-7.10 enzyme. After testing numerous TadA ...10.1038/nature24644 Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, Wilson C, Koblan LW,...

... signals from time to time. False positives in original studies and false negatives in replications are...powered research designs. Unanticipated discoveries Finally, there are good reasons for obtaining a different...replication study. An important difference between the original setting and the new one, previously unaccounted...different handling can all be important.  If we eliminate the problems above: unclear methods, QRPs, and...when replications do not come out the same as an original research finding we can be more certain that some... papers. The precise timing, order, or every combination of materials and treatments is all too often ...reports, the gold standard for testing hypotheses Finally, Registered Reports combine all the best practices...

...anterograde tracer) or upstream from the axon terminals to the soma (retrograde tracer) (Fig. 2). Viruses...only become activated in the presence of Cre recombinase, which is limited to the hippocampus. Thus, only...titers for efficient retrograde transport. For recombinase-dependent vectors, this can cause challenges ...transduction in catecholamine pathways from the spinal cord (Wang, 2018, Metcalfe, 2022) and substantia...impact of this phenomenon is to use a retrograde recombinase-expressing vector (e.g. Cre or FLP) to transduce...projection pathway neurons and then inject a recombinase-dependent chemogenetic vector directly into the...that are physically blocked, such as those in the spinal cord. Now that you understand this new tool, go...

...Originally published Aug 3, 2017 and last updated Apr 6, 2021 by Will Arnold. Introducing NGS in 2017...plexWell technology to easily and quickly create Illumina sequencing libraries. From start to finish the...libraries are then QC’d, pooled, and prepared for Illumina sequencing on our newly donated MiSeq.  For Complete...electrophoretic gel, and qPCR quantification of the final libraries. A broader view of the whole plasmid To...plasmid.     Confirming tags and fusion proteins Finally, we confirm promoters, tags, fusion proteins, and... Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations from Viral... also adds the necessary adapter sequences for Illumina sequencing as well as unique barcodes per sample...

...list of Fragmid destination vectors currently available. Table 1: Fragmid destination vectors Organism...depicted. (Bottom) Schematic depicting available destination vectors for various delivery methods, along with... with compatible inserts. All destination vectors contain an ampicillin resistance cassette and an origin...components you want, simply assemble them into a destination vector via Golden Gate cloning, perform an exonuclease...sequence for every individual assembly. What destination vectors are available? What you’re building ...Organism Delivery Type Destination Vector Mammalian Lentivirus pRDA_512 (RSV-driven; lentiGuide-based...type (lentivirus, AAV, transposon, Plasmid) Destination vector (dropdown options depending on above selections...

...and Shannon Rinaldi in the Addgene lab! Read their Instagram profiles here (Kenkel, Rinaldi)! But leaving...might not be the right choice. I spoke with our Financial Operations Specialist, Lucy Hammer, about why ... have to go back to school right away. Shannon Rinaldi, Addgene’s Lab Manager, was taking classes at her...science, she stuck with it. Lab Manager Shannon Rinaldi values loving what she does. Her parents told her...response to this question came from Lucy Hammer, our Financial Operations Specialist. She said, “I’ve found the...came to Addgene with a goal to learn nonprofit finance, and while I've done that, my big takeaway has ...conversation master knows that although her focus is finance, it is just as important to bring love and fun ...

...and may increase the risk of mistakes or contamination. Finally, choice of concentration method depends...RCL). Replication competence can arise from recombination events during viral production and poses a biosafety...elements across multiple plasmids, the risk of recombination and consequently, the generation of RCLs is ...plasmid. In addition, the promoter of the 5’ long terminal repeat (LTR) is mutated in third generation systems...additional plasmids thereby increasing the number of recombination events that are required to generate RCL. To...volumes accordingly. While you may need to make some final adjustments, this is generally a good place to start... Keller B, Makalou N, Sutton RE. Systematic determination of the packaging limit of lentiviral vectors...

...did not result in significant recombination. If you do see recombination, we recommend ensuring the cells...pooled libraries can lead to issues such as recombination and loss of plasmid diversity, so Addgene strives...strives to distribute aliquots of the original library whenever possible. In this blog, we’ll discuss ... lentiviral backbone might be more prone to recombination, while a library with a large backbone might...diversity of the library is maintained and reduce recombination during the amplification process. In liquid ...factor overexpression library) or is prone to recombination, we recommend amplification on solid media. ...may not have amplification protocols attached.  Final step: validation  Remember, there is no “one-size-fits-all...

...protein tag which can be added to either the N or C terminal of a protein and can inducibly facilitate the ...concentration can yield intermediate protein abundance. Finally, some degron tags can do more than just degrade... the gene of interest and the degron domain is terminal. Between the tag elements (protease and degron...endogenous CRBN E3 ligase complex which will polyubiquitinate the fusion protein. This will target the protein...protein-of-interest and the CRBN E3 ligase complex leading to ubiquitination and proteasomal degradation. Adapted from Nabet...interacting ligands. After an E3 ligase facilitates ubiquitination of the Halo-tagged protein in question, it ...complex members, an E3 ubiquitin ligase complex. Finally, when auxin is added in the form of auxin hormone...