...reprogramming efficiency through the expression of a shRNA against p53, a modified reprogramming factor cocktail, ...are preferred. This is to avoid the chances of insertional mutagenesis, which is the integration of transgenes...disruption of functional genes at the site of insertion and which can potentially promote tumorigenesis... been recorded in past gene therapy trials and remains a safety concern in the clinical field [4]. Three...Antigen-1 (oriP-EBNA-1) system where each vector contains a viral origin of DNA replication, OriP and the... pCXLE-hOCT3/4-shp53-F 27077 OCT3/4 and shRNA against p53 pCXWB-EBNA1 37624 transient EBNA-1   ...Road Ahead." Journal of Alzheimer's disease & Parkinsonism 6.5 (2016). PubMed PMID: 27882265. PubMed Central...

... apply for the position. This post on Business Insider nicely summarizes good job fair practice. Career...get online and take a look at the company or institution’s career page, where you’re likely to find current...conference or job fair, or it may be someone at your institution’s career center. Alternatively, it might even ...other graduate students and post-docs from your institution have gone, and try sending them a professional... professional development, not count on your institution, program, or adviser to do it for you. Expose...if one exists. Get online! Review company and institution career pages. Make yourself visible on LinkedIn... Reading and Outside Resources http://www.businessinsider.com/how-to-get-the-most-out-of-job-fairs-2014...

...diagnosed this year). Research efforts in mesothelioma instead rely upon private donations. Many people think ...However, there is no question that asbestos exposure remains the single-most important factor in developing ...do not work very well. The best option in such instances is palliative care, which helps patients manage...detection techniques and therapies emerge into mainstream use, they will have a much greater impact on ... good detection and treatment get, there still remains the problem of prevention. Asbestos exists pretty... and the president of the organization, Linda Reinstein – whose husband passed away from mesothelioma ...with Senator Barbara Boxer to introduce the Alan Reinstein Ban Asbestos Now Act of 2016 in the U.S. Senate...

...resistant to antibacterial drugs. Resistance can be intrinsic, meaning a basic characteristic that naturally...resistant somehow overtime. One fantastic example of intrinsic resistance would be Mycoplasma species’ resistance...possess cell walls. As a result, Mycoplasma are intrinsically resistant to this class of antibiotic. Acquired...DNA left behind after another bacterium died for instance). These three mechanisms of acquired drug resistance...be unharmed by the compound it produces (think intrinsic resistance), but surrounding microbes would be... time is needed for the drug to work… if the instructions for treatment aren’t followed closely, you could...in the Lab Probe Phage Genomes for Host Binding Proteins Learn How to Use Our Microbiology Pages Additional...

...Singletons in their bins.  The next component of Binning Singletons is when we bring all the bins together at ...invaluable to me as a bench scientist working at an institution where training opportunities weren’t always available...their own labs.  Fried chicken with a side of inspiration  I look back at the meeting with such fondness...for DNA that group similar sequences together in bins, we thought that binning Singletons together would... and time mentors spend with Singletons in their bins. We also sought expertise from many with experience...media. Joe is a lapsed marathon runner, father of twins (AND a singleton), and is proudly "too weird to ...

...alkyne-phenol, to label proteins with an alkyne group for click chemistry reactions instead of biotin — a valuable...locations. C) Enrichment of dual-labeled proteins identifies proteins that had been trafficked from the “source...dual-enrichment and mass spectrometry workflow identifies proteins bearing both labels (which had therefore been ... light, catalyzing the biotinylation of nearby proteins. B) TransitID uses sequential labeling by TurboID...2023 bioRxiv (B-C). They were able to label proteins moving to and from the nucleus, ER, mitochondria...

...frequency of events, instead of relying on clustered dots. In a contour plot, each line contains the same number...machine reads/interprets the signals, the axis is instead divided into positive and negative signal at a ... a ‘gate’; it tells the machine that everything inside the gate is one population and everything outside...which measure light that “scatters” past the cell, instead of fluorescent markers. There are various types...to fluorescent markers and/or cellular dyes and stains. These will be used in a series of one- or two-...different populations using conjugated antibodies against CD11c and CD123. You can see that mDCs are CD11c...

...approach, which focuses on validating antibodies against markers of the human blood leukocyte populations...cases, we would recommend a knockdown approach instead. In this approach, you’re looking for an antibody...permanent knockdown, consider using shRNA vectors instead. With both siRNA and shRNA, it can be difficult...induce/suppress the expression of many related proteins, not just your protein of interest, which can ...interest, which can be difficult to find for many proteins. In practice, it was necessary for us to combine...primary blood leukocytes. The file shared also contains information about the labeling pattern of several...on human blood leukocytes, including antibodies against these markers. The 371 CD markers characterized...

...Kinases: they regulate many proteins, with ~1/3 of human proteins predicted to be phosphorylated on at...swapping of fluorescent proteins and multiplexing is simple when fluorescent proteins with limited spectral...FRET reporters are comprised of two fluorescent proteins with very specific spectral properties that limit... and the orientation of the donor and acceptor proteins, which can be difficult to predict. Slower Dephosphorylation...

...are only expressed when the targeting vector is inserted downstream of a functional promoter (gene traps...2008). While the biology behind this AAV effect remains unclear, it is likely not a coincidence that the...gene. pAAV-SEPT-Acceptor, the preferred vector, contains a promoterless splice acceptor-IRES-neoR gene,... CRISPR-based approaches for the generation of knockins via homologous recombination with a homologous...application - the creation of sequence-specific knockins - for which the AAV approach is most well suited... CRISPR-based approaches for the generation of knockins, it is not always ideal. Because of these limitations...devised methods to deliver CRISPR components as ribonucleoproteins (RNPs) that can be supplied separately from...

...the ability to generate full libraries of mutant proteins. Researchers from the Whitehead lab developed ... Gibson Assembly: An easy-to-use technique that joins homologous DNA fragments in a single-tube reaction...using Gibson assembly is that the longer the oligo insert used, the more likely it will contain random synthesis... enzymes leads to a buildup of uracil and the insertion of uracil into newly synthesized DNA. This labeled...synthesized DNA strand. Each degenerate primer set contains three consecutive randomized bases (NNN) at a ...that the BbvCI enzymes nick the DNA strand that contains their respective recognition site. This means ... matter? Most of the time no, but there are 2 instances where it does. 1) When the backbone vector has...

...plasmid, into which the GOI is inserted, and a Ti "helper" plasmid, which contains Vir genes necessary for T-DNA...gRNA scaffold sequence and allow researchers to insert targeting oligos with minimal cloning required....Dicot Genome Editing Kit from the Stuttmann lab contains a variety of Agrobacterium-compatible, Cas9 containing... T-DNA processing and insertion into the plant genome. B) The Ti plasmid and helper are transformed into...the Vir genes on the helper plasmid and used to insert the intervening region into the plant genome. Created...- promoters used, precise protein sequences or domains, and methods of delivery - the underlying technique...

...reaction that cleaves a nearby RNA sequence that contains a fluorophore that is quenched when intact and...improves sensitivity of the assay. This reaction also contains SSB, strand displacement DNA polymerase, recombinase...fluorescence visualization. In this technique, however, instead of using lateral flow strips to detect the cleavage...Reverse-transcribed RNA samples are amplified by PCR instead of RPA. Unlike RPA, this technique uses Taq polymerase... which is widely available and less expensive. Instead of using the typical PCR machines, the authors ...depend on collateral cleavage by the enzyme and instead relies on its activity on the target regions thus...COVID-19 using CRISPR diagnostics. https://www.broadinstitute.org/files/publications/special/COVID-19%20detection...

...MTRIA for short), where a fluorescent protein is inserted into one of the loop regions of any G-protein-... Figure 1: MTRIA biosensors are generated by inserting a circularly-permuted GFP into an intracellular...calcium reporter Near-infrared (NIR) fluorescent proteins promise better performance for in vivo use and...indicator using small monomeric NIR fluorescent proteins (miRFP670nano and miRFP720) and the Ca2+-sensing...structure-guided engineering of potassium binding proteins, they developed a family of fluorescent potassium...et al. 2022). By fusing two bright fluorescent proteins with different thermal quenching rates (mNeonGreen...Additional resources on the Addgene blog Fluorescent Proteins 101: Fluorescent Biosensors Resources on Addgene.org...

...where they bind to their targets, most commonly proteins. You can then use fluorescent microscopy to look...visualize their target. These antibodies are generated against a specific antigen and then conjugated with a fluorescent...but is not attached to a fluorescent molecule, instead a secondary antibody that targets the primary is...for each primary antibody could get out of hand. Instead, it is more efficient to generate a handful of ... multiplexing is that you can ask how multiple proteins relate to one another in the same sample. For ...see Im, et al. 2019.  Fixation Good fixation maintains sample morphology with minimal impact to the target...spectral properties match your available imaging instruments and the spectral properties do not overlap with...

...studies or make a reporter construct. Fluorescent proteins (FPs) are also the foundation of multiple important...biological research. Photoconvertible fluorescent proteins Newly developed photoconvertible FPs like PsMOrange...repeat the process with these new photoconvertible proteins. With Sattarzadeh et al.’s discovery that GFP ...After exciting GFP-labeled plant cells at 405 nm instead of 488 nm, Sattarzadeh et al. noticed that GFP ...encoding one of the unexpectedly photoconvertible GFP proteins to make a new transgenic line could avoid problems...as well as a wide variety of other fluorescent proteins for your imaging needs. If you need an FP refresher...

...Llamas Required - Synthetic Nanobodies Against Membrane Proteins More resources from addgene.org  Plasmids...single-domain antibodies genetically fused to fluorescent proteins and used to visualize target antigens in living... transgenic mice that express camelid variable domains (aka nanomice), which gives researchers the “best...made to generate the nanomouse. (VHH = variable domains of heavy chain only antibodies) (B) Nanomice express...widely adopted. So thank you to the camelids who inspired this versatile family of tools, the applications...

...and to disrupt endogenous genes at the site of insertion. In both of these cases, transposons combine the...blue) in a plasmid backbone. A separate plasmid contains the transposase gene (red) for expressing the ...sequence is duplicated near the gene of interest insertion site.   The Sleeping Beauty transposon system ...within the genome. The Sleeping Beauty transposase gains its name from the fact that it was recreated from...continues to be altered in order to improve its gene insertion efficiency in primary cells. In 2009, Zsuzsanna...or 5’ end of the gene of interest allowing for insertion in the correct orientation. These plasmids use...

...Carolina (plasmids at Addgene). It consists of two domains, LOV2 and Zdk1, where Zdk1 binds LOV2 with high...normally sits on microtubules to attract other proteins involved in their growth and extension. Now, they...domain. Each half was fused with one of the LOVTRAP domains. When both halves of the modified EB1 protein are...inactivated by the separation of its synergistic domains. This paper demonstrated that the migration of ...fluorophore binding aptamers and MS2-binding fluorescent proteins were the state of the art in RNA imaging in vivo...on the lookout for better, brighter fluorescent proteins (FP) with improved tissue penetrance and live ...

...Argonaute proteins are also present in prokaryotes, and some of these proteins protect against invading...reasons why you might choose NgAgo over CRISPR proteins Cas9 or Cpf1 - keep reading to learn more! Background...: Argonaute and NgAgo In eukaryotes, Argonaute proteins play an important role in RNA interference, binding...when the protein is being synthesized, so NgAgo remains faithful to its guide DNA and cannot switch guides...

...usage has been limited by technical problems. For instance, it’s much more difficult to introduce a gene ...strategy for transducing primary cells. What if, instead of supplying a DNA construct, they could simply... adapted from D’Astolfo et al. In certain instances, the potential benefits of nonviral transduction...with virus work. Fourth, working directly with proteins removes the limitations of packaging size associated...References Efficient Intracellular Delivery of Native Proteins. D'Astolfo DS, Pagliero RJ, Pras A, Karthaus WR...PubMed. Find the Plasmids @Addgene: iTOP-tested proteins and reporters Previous Protein Delivery Techniques...

...Tom, and Alan E. Tomkinson. "Eukaryotic DNA ligases: structural and functional insights." Annu. Rev. Biochem...many molecular biology protocols, including vector-insert joining for recombinant plasmid construction (restriction...DNA ligase, are naturally able to discriminate against ligating substrates containing base pair mismatches... can be achieved through a combination of the intrinsic selectivity of the ligase active site and careful...indicated that the optimal buffer for Taq DNA Ligase contains 100 – 200 mM KCl at pH 8.5. This optimization....6 (2004): 757-764. PubMed PMID: 15582400. 2. Tomkinson, Alan E., et al. "DNA ligases: structure, reaction...

...freeze-dried, cell-free (FD-CF) components. Making proteins outside of a cell - cell free extracts Typically...transformation), essentially giving the cell all the instructions it needs to produce that green fluorescent protein...systems contain all the parts needed to produce proteins without the need to handle a living, replicating...simply need to add water to express a variety of proteins. FD-CF reactions are also cost-effective, as each...easily sense, whether that’s bright fluorescent proteins that they can see, enzymes that can convert certain...activities fit your lesson plans. If you are an instructor at the college level, perhaps consider using ...

...accumulate inside the producer cells and purification protocols often describe a process that begins with cell... carried inside the vesicle, which may also contain other cellular molecules such as proteins or nucleic...cells also contain cellular macromolecules (e.g. proteins, nucleic acids) which possibly improve transduction... contexts, such as the delivery of therapeutic proteins to the brain which requires crossing the blood-brain-barrier...

...but can also be used to brainstorm on data in smaller groups, where for instance the presence of the PI...lab meetings with a focus on discussion and brainstorming Though, we might be forced to work from home...of each talk - time for free interaction and brainstorming. This could be achieved by asking each presenter...problem! You can still creatively collaborate and brainstorm! Image from Joseph Mucira.   Implement new... be written by several authors from different institutes, disciplines, and cultural backgrounds.” This...

...confirm the presence of an insert in a particular vector by excising the insert from the backbone. To do...use enzymes with restriction sites that flank the insert. You will need to know both the approximate size...vector backbone as well as the predicted size of the insert. You can search NCBI for YGOI to find the particular...right has a total size of 7.3kb, including a 1.2 kb insert. The plasmid was digested with 2 unique enzymes... and 1.2kb (red box), matching the backbone and insert, respectively. The results on the gel correspond...

...Associate Director of the Cancer Program at the Broad Institute of Harvard and MIT. The notion of cancer precision...We've developed a new plasmid toolkit, the Broad Institute Target Accelerator Plasmid Collection, that we...variant function and cancer phenotypes. Broad Institute Target Accelerator Plasmid Collections To overcome...this first challenge, we’ve developed the Broad Institute Target Accelerator Plasmid Collections, now widely...Associate Director of the Cancer Program at the Broad Institute of Harvard and MIT and is focused on making precision...Kits at Addgene Browse Plasmids from the Broad Institute ...

...Root from the Broad Institute and Alex Chavez from the Wyss Institute were instrumental in helping us establish...School, and Jonathan Ting’s lab from the Allen Institute have supported the AAV initiative from the beginning...Janelia Research Campus have provided valuable insight into AAV production. Finally, we are blessed to...permission from relevant plasmid depositors and their institutions to distribute viral preps derived from plasmids...

...restriction enzyme cloning uses short sticky ends to insert genes into plasmid backbones, base pairing between...absence of the other dNTPs. Once the vector and insert are (separately) digested to create 10–12 base ...on the power of homologous recombination. SLIC inserts and linearized vectors must have homologous DNA...imprecise T4 digestion (no dNTPs needed), allows both insert and vector to be digested in the same tube. Alternatively...these approaches require higher concentrations of insert to succeed. 5′ overhangs are generated in the linearized... be used with different types of PCR-generated inserts. By harnessing the power of DNA repair, you can...

...interact with the host. This is the role of Gag proteins: they contain the matrix, capsid, and nucleocapsid...assembly, and display. Pol, the last component, contains the reverse transcriptase and integrase components...convert the cargo from RNA to DNA and permanently insert the cargo into the host genome. Lentiviral-specific... Viral packaging and entry The viral packaging proteins for AAV are rep and cap. Rep is required for viral...discussed. LXs/Iva2 are primarily capsid assembly proteins required for packaging of the virus into particles...Resources Kulanayake, S., Tikoo, S. K. Adenovirus Core Proteins: Structure and Function. Viruses. 2021 13(3):388...

...proto-oncogenes and disrupt their expression (Robinson, 1982). To add insult to injury, retroviruses generally have...This viral DNA (also called proviral DNA) is then inserted into the host's genome and is used to make more...virus-specific elements such as the LTRs or structural proteins are not interchangeable between the two systems...Adenovirus generally has high immunogenicity, but some strains have been engineered to induce a lower inflammatory...immunogenicity low! References and Resources References Robinson HL. Retroviruses and cancer. Rev Infect Dis. 1982...

...if not methylated (if it comes from a phage for instance). Aside from restriction modification systems,...effectively cleave DNA. Common lab E. coli K12 strains such as DH5alpha contain 3 methylases that recognize...Dam or Dcm methylation, you can ensure this site remains unblocked by first cloning your DNA into a dam–...and re-purifying it. These specialized E. coli strains have been specifically engineered to be Dam and...those sites. Please keep in mind that dam-/dcm- strains may have an increased rate of mutation (as these...in mis-match repair functions of Dam), so these strains should not be used for long term storage. Additional...

...another name for a folder. For instance, your Desktop directory contains the folders and files that you...including background on other analyses that have inspired you to perform this analysis. Having a clearly...multiple projects in a given day. For example, instead of naming a file “output” name the file “1_output...subsequent word in a file name after the first word begins with a capital letter and words are not separated...temporarily used. Just be sure that the original code remains in tact! Take notes Comment in your scripts: ...

...small percentage of the proteins inside a cell have a commercial antibody against them. There’s also a question...Mendenhall of the University of Alabama in Huntsville explains it, a major goal in his laboratory is to understand...track down every last one of those gene regulatory proteins and the places in the genome where they can be.... If things work correctly, the second plasmid inserts a small epitope tag called FLAG at the end of the...

...and control genes, but according to the Broad Institute's Silvana Konermann, the first generation of CRISPR-Cas9...recognized as an ideal spot to fuse the activators instead of at the protein’s C terminus. With that new design... and knew she was on the right track. The next insight was to assemble a synthetic transcription activation...complex consisting of multiple distinct effector domains that work together synergistically, modeled after...CRISPRa) sgRNA library uses the sunCas9 system and contains 10 sgRNAs for each transcription start site in...The transcriptional repressor (CRISPRi) library contains 10 sgRNAs for each transcription start site in...

...occurring resistant mutants and the insensitivity of yeast strains to some antibiotics. Auxotrophy is ...bacteria, yeast can post-translationally modify proteins yet they still share many of the same technical...replication (Please note: This first section primarily pertains to ORIs in budding yeast, Saccharomyces cerevisiae...other hand, do not require a well defined ORI. Instead, the size and A-T content of the DNA (apparently...compound required for its growth. Many auxotrophic strains of yeast exist which can be easily maintained when...

...multiplexing of sgRNAs against unique genomic targets or multiple sgRNAs against the same target to ensure...protein purification and metabolic engineering. If instead you want to build genetic circuits and would benefit...library of parts but don’t need to add tags to your proteins, then the CIDAR Kit is an excellent option (Iverson...system includes a toolkit to express recombinant proteins within the mitochondrial matrix.     Figure...can be repurposed for CRISPR applications (gene insert = Cas9 + gRNA), Addgene distributes specialized...of a Toolkit for the Expression of Heterologous Proteins in Yeast Mitochondria.” BMC Biotechnology, vol...

... ZIF268 DNA binding domains fused to either the hER or hPR ligand binding domains and the MSN2 transactivation...Schuh labs. Clift et al. applied TRIM-away to nine proteins in ten ddifferent cell types, including difficult-to-manipulate...TRIM-Away is suitable for both short-and long-lived proteins, and it can even distinguish between protein variants... STED nanoscopy Article contributed by Eric J. Perkins Listen to Endosomal pH Sensors podcast segment ...plasmids encoding fluorescent reporters and other proteins controlled by GAL4 and ZIF268. To demonstrate ...

...The Rare Genomics Institute BeHEARD (Helping Empower and Accelerate Research Discoveries) Challenge provides...our fourth year working with the Rare Genomics Institute by providing plasmid-based tools for rare disease...Addgene to set up assays to see what molecules or proteins are involved in IL-36 release in keratinocytes... valve (an aortic valve that has two leaflets, instead of three) and acute aortic dissections (a tear ...standing interest in the biology of GPCRs and G proteins, with an emphasis on oligomerization, signalling...Resources Find out more about the Rare Genomics Institute and this year's BeHeard Award! ...

...genomic deletions with or without small insertions. But, instead, PEDAR couples this deletion process to...mammalian cells, especially when fused to effector proteins for gene activation or gene editing. The more ... lab adapted the native PEG10 RNA sequences to instead drive packaging of cargo sequences in the capsid...it is also capable of coding concurrent small insertions to allow in-frame deletions and introduce epitope...mouse model of tyrosinemia, where a pathogenic insertion within the FAH gene was precisely corrected, restoring...

...plasmid is the correct expected size and contains the correct insert Sequence the regions that have been amplified...DNA. As a general rule, try to use an excess of insert compared to the backbone plasmid; a starting point...point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized according...transforming the ligation mix in different bacterial strains (stbl2, NEB stable, etc.), and/or use a different...primers. If not, design primers to sequence your insert. When you get your sequencing results back, you...

... learned over the years with the hope that our insights can help steer you in the right direction when... in our current setup, there are no explicit instructions on what to do when something fails. While we... through which she could easily transfer her instructions for dealing with the samples to newer lab members... ourselves up for success by giving explicit instructions to lab members through the software if there...collisions you may encounter: vector, sequence, insert, trait, factor, expression, etc.There are also ...biology terms (plasmid, 5-prime, CRISPR, growth strains, vector backbone, etc.) and you simply don't have...

...As Christopher Voigt explains it, his lab at the Massachusetts Institute of Technology has been “working...create an almost limitless number of DNA binding proteins with Cas9 guided by different promoters. The challenge... genetic circuits is getting enough regulatory proteins that don’t interfere with each other. CRISPR is...much easier it is to work with than regulatory proteins. There are still things we haven’t solved that...Nothing too much more sophisticated. There are also proteins, like antibodies. But we are really just using...

...controlled by deliberate and compulsory actions instigated by the central nervous system signaling to peripheral...sensors for a diverse set of fat-soluble hormones, vitamins, and dietary lipids. Included in this family are...progesterone, androgens, and estrogens), fat-soluble vitamins A and D, thyroid hormone, fatty acids, oxysterols...composed of a tightly-bound invariant group of proteins, whereas the more loosely bound co-coregulators...explain how more than 300 different coactivator proteins individually contribute to cell regulation in ...acetyltransferase to luciferase, the basic assay remains widely used today.  The sheer abundance of these...

...or non-existent PAM requirement, and how these proteins are advantageous in today’s genome editing landscape...the site in question (Kwart et al, 2017). To add insult to injury, the most utilized PAM sequence, that...requirements. Luckily, there are some available.   Cas proteins that don’t utilize NGG   There are plenty of Cas9...same PAM preference of NG was isolated by the Kleinstiver Lab (read more on this lab's other developments...developments below!) (Walton et. al, 2020). These Cas proteins have everything we know and love about Cas9, but...and location.   Nearly PAM-less editing   The Kleinstiver lab sought to address the PAM problem through...

..., and Cas11. Type I-E Cascades contains all five of these Cas proteins, while type I-C Cascades do not...boundaries defined by anti-CRISPR proteins or running into other proteins naturally bound to the DNA. Cas3...The helicase domain is then activated, and Cas3 begins unraveling the double-stranded DNA (dsDNA) in what...Cas9 vs. The Other Cas(s) CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems INTEGRATE: Bacterial...

... as “instillation” or “gavage,” as they do not require any punctures. Intratracheal instillation Intratracheal...Skill required: advanced Intranasal instillation Intranasal instillation is very similar to its intratracheal...compounds, as circulation is slower, and more institutional oversight is required. Restraint: mechanical...Intratracheal instillation is used to deliver substances directly to the lungs. It is an advanced technique...

... depending on your flow cytometer’s setup. For instance, BV510 and AmCyan have almost identical emission...and how do I fix it? When plotting two channels against each other, you should be able to draw perpendicular...single-color APC control, with APC and APC-CY7 plotted against each other. Since it is a single-color control,...After increasing the compensation values for APC against APC-Cy7, the populations are in more of a straight...compensation value of five was applied to APC-Cy7 against APC to correct for this bleed. A compensation value...population is not a double-positive population and is instead due to bleeding, you will see the populations move...

...resistance selection markers, gRNAs, and dCas9 proteins. Second, it can be introduced into a broader range...tnsB, tnsC, tnsD, and tnsE), and a plasmid that contains the Mobile CRISPRi cassette flanked by left and...recipient's genome. Antibiotic plates lacking DAP select against donor bacteria and for recipients with an integrated...CRISPRi to non-model bacteria including pathogenic strains and environmental isolates. For example, Peters...expressed RFP reporter, was variable across bacterial strains, ranging from an ~8 to 150-fold reduction, with...the relative frequency of each of the 40 library strains was measured after 6 and 12 generations with or...

... myself writing AAV production tricks into the margins of my protocol. While these tips weren’t critical...replication uses the host’s polymerase, but requires Rep proteins to process a double-stranded intermediate into...cultures of your AAV transfer plasmid at 30 °C instead of 37 °C and then screen for ITR recombination ...transform AAV transfer plasmids into bacterial strains, like NEB Stable. NEB Stable competent cells lack... should consider performing PEG precipitation. Instead of sonicating the cell pellet, I lyse the cells... Stone and Harshana de Silva Feelixge at Fred Hutchinson Cancer Center for teaching me about AAV production...