... 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 ...

... 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...

...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...

...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,...

...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...

... 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...

...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...

...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 ...

...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...

...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...

...unique repair templates. A final ligation reaction correctly assembles the final targeting vector (Fig. 1...need for PCR or cloning, as digestion of the destination vector with SapI yields 2 sites- the first site... the same unique SapI 5’ overhang, different combinations of connector and tag donor plasmids from the... a tissue specific manner, as well as 3-site destination vectors for inserting a tag at multiple target...the Hornung Lab, allows users to easily create C-terminal tag fusions of endogenous genes in human cells...plasmid containing the homology arms and an N- or C-terminal tag. The two PCR reactions are then mixed and ...expressing Cas9. This PCR toolkit offers C- and N-terminal tagging vectors (eg GFP, Flag, YFP, Strep, TEV-V5...

...researchers test many different combinations of promoters, terminators, solubility and purification tags...connectors, cDNAs, and more to find the perfect combination. The EcoFlex Kit has a wide array of DNA parts...EcoFlex a great option for those interested in recombinant protein purification and metabolic engineering...expression, the system includes a toolkit to express recombinant proteins within the mitochondrial matrix.   ...specialized system with parts including site-specific recombinases, regulatory elements, developmental regulators... examine the effects of different promoters, terminators, enhancers, and cross-cassette interactions in...localization and transport studies in HEK cells, recombinant protein purification in bacterial cells, and ...

...Cas13a: An RNA cleaving CRISPR nuclease Cas13a was originally identified in 2015 (Shmakov et al., 2015). They...enzymatically “active” state rather than reverting to an inactive state like Cas9 or Cpf1. As a result, Cas13a will...sequence of interest (red) is amplified using Recombinase Polymerase Amplification (RPA) or Reverse-transcription...crRNA targeting the sequence of interest and an inactivated fluorescent RNA reporter. If the target sequence... whether the target sequence is present in the original pool of nucleotides.   What are the potential...regulating expression of specifically targeted RNAs. Finally (and perhaps most interesting), Cas13a’s propensity...-targets. They subsequently used catalytically inactive dLwaCas13a as a programmable RNA-binding protein...

...presence of that metabolite (Fig.2). Spinach eventually evolved into Spinach2, a more stable and brighter version...essentially mimicking GFP.  Though the aptamer, called Spinach, required longer exposure time than its protein...possibilities. For instance, further selection of Spinach with a target metabolite creates an aptamer that...but it still had limitations in vivo. Using a combination of SELEX and fluorescence-activated cell sorting...also has a higher melting temperature than its Spinach ancestors, and has a lower magnesium dependence...Chemistry 27(2):108-117 · February 2008. 11. Svensen, Nina and Jaffrey, Samie R. “Fluorescent RNA Aptamers ...Activity with GFP Resources at Addgene.org Find the Spinach Plasmids at Addgene Find Broccoli Plasmids at Addgene...

...hinders experiments is mycoplasma contamination. Mycoplasma contamination has been shown to arise from a ...Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention... growth and behavior. Signs of a mycoplasma contamination can be subtle and may include reduced saturation..., labs should routinely test for mycoplasma contamination. There are several commercially available mycoplasma...PCR-based approaches to detect even low levels of contamination. For those labs that prefer a more economical....C., Drexler, H.G. “Detection of mycoplasma contaminations.” Methods in Molecular Biology 946 (2013). ...

...coli strains are not usually well suited for recombinant protein expression. Many challenges can arise...coli. We will review the potential pitfalls of recombinant protein expression and some of the most popular...or are enzymatically active. For this reason, recombinant proteins are typically expressed in E. coli engineered..., which reduces proteolysis of the expressed recombinant proteins. lon protease: Strains where this is...proteins. hsdSB (rB- mB-): These strains have an inactivated native restriction/methylation system. This means...plasmids utilize inducible promoters, which are 'inactive' until an inducer such as IPTG is added to the...to make protein.  The DE3 lysogen/T7 promoter combination is the most popular induction system. The DE3...

...validation data, or follow the references to find the original publication and ensure that it includes validation...-lot. The highest reproducibility comes from recombinant antibodies (rAbs), which are produced from plasmids...plasmid sequencing. While rAbs are monoclonal, recombinant multiclonal antibodies — mixtures of different...re-purchasing your primary antibody with the new reporter. Finally, each new conjugate will have to be tested to ensure...It is recommended for small to medium proteins. Finally, there are dry/electroblot transfer systems available...range, they cannot be read with equal accuracy. Finally, they degrade quickly and need to be read fairly...compatible with stripping and probing.   Imaging At the final step of your western, you’ll have two choices: x-ray...

...dietary supplements Dr. Mrinalini Nair, University of Baroda, India Dr. Mrinalini Nair, Associate Professor... viruses. Now focusing on plant biology, she coordinates the Network for the Genetic Improvement of Cowpea... order to manage insect pests and to support sustainable agriculture. For this project the expression ...LIC cloning vector and the pOKD plasmids. The recombinant Cry proteins will then be screened for toxicity...oxidase. The goal is to determine potential water contaminants and to subsequently develop technologies to ...who applied, and to Seeding Labs who helped us coordinate and promote the awards. References 1. Martinez-Klimova...

...AAV5 Recombinases Stanley Thomas Carmichael New viral prep pAAV-EF1a-fDIO-Cre AAV5 Recombinases Esteban.... These preps drive expression of either Cre recombinase or a fluorescent marker in the various cell types...serotype pAAV-hSyn-Cre-P2A-dTomato AAV PHP.eB Recombinases Rylan Larsen New serotype AiP13778 - pAAV-...pAAV-AiE0450h-minBG-iCre(R297T)-BGHpA (Alias: CN3778) AAV PHP.eB Recombinases Jonathan Ting New viral prep AiP13779 - pAAV-AiE0779m...minBG-iCre(R297T)-BGHpA (Alias: CN3779) AAV PHP.eB Recombinases Jonathan Ting New viral prep AiP13781 - pAAV-AiE0452h-minBG-iCre...pAAV-AiE0452h-minBG-iCre(R297T)-BGHpA (Alias: CN3781) AAV PHP.eB Recombinases Jonathan Ting New viral prep pAAV-CAG-GFP ...

... mountain – with many routes up and only one destination – a career in the sciences is more like an intergalactic...a simple egg is packed with information. I am fascinated with how developmental programs unfold, absorbing...comparative anatomy showed me that some of the most fascinating biology is found in weird and rare organisms ...stick to vertebrates alone.  And a longstanding inclination to be a teacher was looking for an outlet. I ...Association for Women in Science); others were visiting seminar speakers. My eye was caught by a couple of unusual...how punctuation can be wildly funny as well as financially critical. Those who work on the publishing side...the right audiences and how open access affects financial models in information publication. Take a look...

...produce recombinant proteins at high levels. In a case where clinical applications of recombinant proteins...wondered where human embryonic kidney (HEK) cells originated from? Or why they are such a commonly used cell...difficult to determine its true tissue origin. Originally, it was assumed HEKs were a common cell type ... necessary, HEKs are human in origin, so they eliminate non-human PTMs which is a concern with other protein...cultured human cell lines are susceptible to contamination by mycoplasma and bacteria, but HEKs are also...., HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors. Front Bioeng Biotechnol...

...conductor receptor (CFTR) by homologous recombination in cultured intestinal stem cells of patients with cystic...This post was originally written in 2014 by Kendall Morgan and updated in 2022 by Lucie Wilson. Lucie...CRISPR/Cas9 could be used to edit several autosomal-dominant mutations that have been connected to the early...Chinese Academy of Sciences found that mice with a dominant mutation in a gene that causes cataracts could... chose to explore its potential in mice with a dominant cataract disorder caused by a single copy of a... "Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients...

...interest: The transgene sequence is flanked by long terminal repeat (LTR) sequences, which facilitate integration...line that stably expresses Gag-Pol and/or Env, eliminating the need to deliver these genes in trans by specific...feature may be useful for the transduction of terminally differentiated (non-dividing) cells. Both lentiviruses...produce additional virus. Are γ-retroviruses self-inactivating (SIN)? They can be. Plasmids that encode SIN... LTR Subcomponents: -U3 -R -U5 LTR; Long terminal repeats; U3-R-U5 regions found on either side ...U5 and the rest of the provirus. 3’ LTR Terminates transcription started by 5' LTR by the addition...written and updated by multiple Addgenies. It was originally published in 2018, in the Viral Vectors 101 eBook...

...pAAV-hSyn-DIO-hM3D(Gq)-mCherry Bryan Roth   Recombinases Recombinase systems such as Cre-lox allow for more...types, recombinases allow you to further restrict gene expression to areas in which the recombinase and ...and construct are coexpressed. Three new recombinases from the Cepko Lab are now available as AAV1. CRE-...constructs 69570 and 69571 together encode the N and C terminal segments respectively of a GFP dependent Cre. ...to form a functional Cre enzyme that induces recombination of lox sites. This system takes advantage of...