...confirmation of recombinant clones If a antibiotic resistance gene has been inserted, recombinants can first...major advantage of using this method for lambda red expression is that it doesn’t require antibiotic selection...  References 1. Costantino N, Court DL. Enhanced levels of λ Red-mediated recombinants in mismatch repair...outgrowth of bacteria. Selection and confirmation of recombinant clones.    Figure 2: Overview of using... system to replace a gene of interest with an antibiotic resistance cassette.   Substrate DNA Design..., including selectable DNA fragments, such as antibiotic resistance genes, as well as non-selectable DNA...replacements and tags.  The typical frequency of recombinants is 1 positive clone out of 104 to 105 colonies...

...utilization, Arabidopsis has made significant contributions to understanding plant and crop science alike.  Arabidopsis...for use in Arabidopsis. There are also important tips for plant transformations and the use of Arabidopsis... The development of Arabidopsis as a model plant. The Plant Journal, 61(6), 909–921. https://doi.org/10.1111...page Find more plasmids and resources for plant resources on Plant Plasmids Collection Page Read our molecular...Justin Ng and Alyssa Cecchetelli Science is a constantly evolving and demanding field, requiring a variety...vastly expanded axolotl research. One of the most significant advances is the sequencing of the entire axolotl...research providing insight into some of the most significant and fundamental biological processes. Many of...

...site-directed mutagenesis.  Quantitative polymerase chain reaction, qPCR Quantitative polymerase chain reaction... to quantify the concentrations of multiple starting DNA templates in one sample. It is important to note...heating and cooling steps. PCR relies on heat resistant DNA polymerase from the thermophilic bacterium...aquaticus (Taq). Taq polymerase is thus a heat resistant enzyme that can withstand changes in temperature...to confirm product presence, size, and relative quantity. End point PCR is most commonly used in molecular...genotyping. It’s extremely useful but is not as quantitative as other methods of PCR. Theoretically scientists...scientists should be able to determine the quantity of DNA after a PCR reaction as the amplicon doubles every...

...substrates/peptides and antibodies. Example applications of this include coupling to an antibody, whereby excitation...agonists and antagonists, impacting the interpretation of experimental results. It's important to consider...required. These dyes can also be attached to an antibody to detect various targets, such as proteins, nucleic... These consist of a genetically encoded enzyme variant tag that covalently reacts with a small substrate...through fluorescent labeling. There are several advantages to using these self-labeling proteins over traditional...near-infrared fluorophores. Fluorescence microscopy takes advantage of the characteristics of these fluorophores to...protein function and cellular components: It's important to assess how dye-conjugation affects protein ...

... you want to use optogenetics The most important question to consider is what exactly do you want to use...and components necessary to run it? And, most importantly, what are the methodological and experimental...considerations? I recently spent two months in the lab of Antonello Bonci, Director of the National Institutes of ...optogenetics for? The technique is non-trivial; you don’t want to spend months doing surgeries and making fiber...region you wish to stimulate 2) Fabrication and implantation of the optical fiber into the region of interest...any optogenetic experiment. There are numerous variants for the different types of opsins available and...of North Carolina) that offer his constructs. Importantly, control viruses (not expressing the opsin) are...

...plasmids, multiple Cas9 variants are available: wt humanized Cas9, D10A nickase mutant (Cas9n), dCas9 (transcriptional...pCAMBIA. Cas9 is plant-optimized, and plasmids are available for both monocot and dicot plants, with a choice...Cas9 toolkit for multiplex genome editing in plants.” BMC Plant Biology 14 (2014): 327. PubMed PMID: 25432517...multiplex genome editing in monocot and dicot plants.” Molecular Plant 8(8) (2015): 1274-1284. PubMed PMID: 25917172...suitable for other model systems, including E. coli, plants, Drosophila, and zebrafish!   Why use multiplexed...This increases the chance that all the edits you want to make will happen.  There are many reasons to ...with Golden Gate and Gibson assembly methods If you want to scale up the number of gRNAs in your plasmid,...

...expressing the recombinant plasmid to propagate. Any cells that contained non-recombinant vectors (re-ligated...DB3.1, which contains a mutant version of DNA gyrase (gyrA462) that is resistant to the toxic effects of...factor plasmid of E. coli, is part of a toxin-antitoxin system encoded by the ccd operon, which is responsible...another gene found in the ccd operon, codes for the antitoxin protein (CcdA) that protects the cell against ...the number of clones that do not contain the recombinant plasmid and therefore makes the cloning process...modern version of this older system, with added advantages that will be discussed in detail in a separate...post. Focusing on the ccdB aspect, Gateway takes advantage of the same principle that cells will not propagate...

... develop a way to quantify the take rate of mouse xenografts after cell implantation. Their goal was to...cells that represent the preexisting resistant subpopulation 2. Resistant cells among the separate replicates...ClonTracer library, Bhang et al. found that antibiotic resistant cancer cells exist prior to drug treatment...majority of the resistant populations. In fact, 88% of the barcodes within the resistant populations were...decided to do a post-doc at Novartis because she wanted first-hand experience in industry research. When...determine whether or not cancer cell populations resistant to drug treatments arise during treatment or are... outcomes (see schematic depiction below): 1. Resistant cells among the separate replicates would contain...

...Jordan Ward who is an assistant professor at the University of California, Santa Cruz. Emerging CRISPR...blogger! Jordan Ward is an assistant professor at the University of California, Santa Cruz where his group ...CRISPR/Cas9 editing strategies in C. elegans As brilliantly and irreverently illustrated in the parable of...fluorescence (GFP; Tzur et al., 2013), or rescue of mutant phenotypes (unc-119) (Dickinson et al., 2013). ...Andy Fire’s lab used oligo-mediated knock-in of a dominant mutation, known as co-conversion (Arribere et ...-CRISPR and co-conversion approaches have the advantage of being used in any genetic background, but require...repair of a temperature-sensitive pha-1 mutation significantly enriches for knock-in of 2x and 3xFLAG epitopes...

...travel, teaching, grant writing, etc. Also, it is unlikely that a mentor has the only relevant expertise and... phenomenon? Exploration can be important, but it is equally important to have a plan to get to mechanism...talk takes significant effort. It requires evaluation of all of the data and any recent relevant studies ... often more easily done in reverse. Assuming you want to become competitive for a Group Leader position...preparation) A history of successful funding (fellowship grants) A project with sufficient preliminary data to...your own lab and get an independent investigator grant (e.g. an R01 from the NIH, in the United States)...researcher. Depending on the position, you may also want some evidence of teaching experience. How do you...

...team, you don’t necessarily want a lot of redundant skills - you just want the necessary skills. You might...but equally important is making sure all team members understand what roles they’re meant to fill and ...equivalent of a senior thesis), I knew early on that I wanted to work with a partner. WPI’s emphasis on teamwork...year isolating pheromones from various C. elegans mutants by myself, I would have slowly gone crazy. As it...and others, we have updated our kit process substantially and have plenty of ideas for further improvements...year, or a few months, or even a few weeks. The important thing to remember is that every team should have...when I do come across one. Also, remember, one important feature that all projects have in common - and...

...weekend - why? Do they have an important plans? What is most important to each person? Someone has to... in the area I want to explore while also ensuring that my work gets completed? I want my company to pay...everyday What I didn’t realize was how much I was constantly negotiating, every day. As a grad student, I ...I was negotiating with my PI on how to spend my grant money (Can I attend this conference?) and with my...negotiation as a zero sum game - that is, if I get what I want, that means you don’t. In this mindset, one of us...big conference - Is one of those options more important to you and why? What are their expectations/...someone doesn’t collect their data), ask why you both want this time and what your needs actually are: Maybe...

...of Adeno-Associated Viral Vectors by Oxidant-Based Disinfectants. Human Gene Therapy, 32(13–14), Article...basics of rAAV production.  Of course, you will want to make sure that your final stocks only contain...your cells, wait 2–5 days and then collect the supernatant and/or producer cells for further processing....depend on the serotype, as some rAAV serotypes are abundantly released into the culture media, while others...you can consider using unpurified AAV from the supernatant (Goodwin et al., 2020) or crude cell lysate (...  Purification To purify your rAAV from your supernatant, you can simply use polyethylene glycol (PEG)... 25 mL of 40% PEG solution to each 100 mL of supernatant (final concentration of 8%). If, however, you...

...the acquisition of antibiotic selectable markers by the destination plasmid. Antibiotic selection allowed...suppression of background by antibiotic selection. Screening of transformants by colony PCR confirmed the...number of transformants); the greater the number of inserts, the lower the number of transformants. To compensate... by guest bloggers Becky Kucera, M.Sc. and Eric Cantor, Ph.D. from New England Biolabs. Golden gate assembly...Lambda insert at the same high frequencies as the antibiotic selection markers. The 5-, 12- and 24-assembly...larger volumes of the outgrowth containing the transformants onto the selection plate. Figure 2 shows representative...assembly was easily achievable with high levels of transformants and low backgrounds – so much so that there ...

...not to mention frustrating. One elegant solution to sidestep the antibody problem is to tag the protein ...Bressan RB, Dewari PS, Kalantzaki M, Gangoso E, Matjusaitis M, Garcia-Diaz C, Blin C, Grant V, Bulstrode H, ...Regenerative Medicine (CRM), Edinburgh. Most commercial antibodies do not work in pull-down assays: Epitope tagging...protein complexes - from the cell-lysate using antibodies against the target protein. The captured protein...to find (or generate in-house) a pre-validated antibody for each target protein. To study 20,000 human...immunoprecipitated using a standard tag-specific antibody (Jarvik and Telmer 1998). Indeed, this method ... of interest, and a universal all-purpose “tag antibody” can be used for downstream IP/ChIP assays. Creating...

...photostability, quantum yield, and expression. Create yellow (v82) and lime green (v857) variants differing ...for sfGFP to create SF-iGluSnFR. This improved variant has higher expression levels in bacteria and produces...models. Furthermore, they created three affinity variants – A184S (increased affinity for glutamate), S72A...Kaspar Podgorski’s lab have recently made iGluSnFR3 variants that are more sensitive and better localized for...predecessors. The detailed characterization of the new variants is available here. Upgrades to iGluSnFR3 To improve...kinetics, affinity, and ∆F/F0. Introduce the two variants in three different membrane-targeting flavors ...Improved photophysical properties The improved variants of iGluSnFR3 have higher expression levels and...

...' TTTV AsCpf1 RR variant 5' TYCV LbCpf1 RR variant 5' TYCV AsCpf1 RVR variant 5' TATV Campylobacter... PAM sequence. Cas9 Species/Variants and PAM Sequences Species/Variant of Cas9 PAM Sequence ...NGG SpCas9 D1135E variant 3' NGG (reduced NAG binding) SpCas9 VRER variant 3' NGCG SpCas9...SpCas9 EQR variant 3' NGAG SpCas9 VQR variant  3' NGAN or NGNG  xCas9 3' NG, GAA, or GAT SpCas9...general overview of CRISPR components, new Cas9 variants are being discovered all the time and the requirements...different systems can vary (for example, xCas9 is a variant with increased PAM flexibiliy and eSpCas9/SpCas9...protein required is the Cas9 endonuclease or a variant thereof. This individual protein has all the components...

... histogram plots to quantify smeary plots For smeary expressions, you can quantify shifts in signal using... cytometer and see one of two results for your antibody of interest: two clear populations or a huge smear...post, I’ll walk you through how to interpret the antibody readout on a flow plot.  Note that for the flow...performed. Before you read out your plots, you’ll want to make sure you’ve also adjusted voltage and compensated...cleaner readout. A yes-no plot If you are using an antibody to mark the presence or absence of a protein, ...anything to the right of it, marking it as GFP+. Quantification for such graphs could simply be the percentage...Figure 2: (A) a smeary dot plot showing a range of antibody readouts. (B) A gate on the smeary dot plot shows...

...aims do not need to be grant aims. Each aim could be the basis of a single grant. Either way, think realistically...For example, “there is a significant need for healthy livers for transplants. Growth of new livers in .... Given this large sum, one might expect that applicants would work for months to hone a chalk talk. Yet...talk could be your plans for your first NIH R01 grant proposal or a 10-year research plan. I suggest preparing...research vision is your approach to resolving a significant knowledge gap in your field or even creating ...vague goal or a "what" question. For example, "we want a better understanding of the Z pathway" is vague...techniques that can help resolve the question, the anticipated outcomes, the alternative approaches, and what...

...So the development of near-PAMless Cas9 variants significantly expands the library of accessible genomic...Kim et al., 2017 SaBE3, BE3 PAM variants, BE3 editing window variants   Greatly expands the number of ...processes. BER = base excision repair. Note: hypoxanthine (shown) is the nucleobase component of the nucleoside...increase base editing efficiency, Komor and team wanted to force the cell to use the deaminated DNA strand...upstream of the PAM.  David Liu’s lab generated BE3 variants with other deaminases: AID, CDA1, and APOBEC3G... Liu lab also used natural and engineered Cas9 variants to develop five new base editors with distinct...HF-BE3, a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017). HF-BE3 showed 37-fold...