...for Host Binding Proteins & Running for Rare Disease, Running for FOP, Running for AJ I have two picks for...the post. The second post, “Running For Rare Disease, Running for FOP, Running for AJ,” was contributed ... we’re raising money for a cause while running. I love running and this event gives me the opportunity...sitting in the San Francisco International Airport listening to the Lion King soundtrack and writing this post...Project That’s it for now. As I indicated in the opening paragraph, I’m currently on my way back from a ...

...generate gRNAs and homology arm repair templates containing the tag for each insertion. Also, it remains ...simultaneously introduce a selection marker for screening modified strains. With SapTrap, the user first... (Fig. 1, Step 1). There is no need for PCR or cloning, as digestion of the destination vector with SapI...SapTrap includes a prebuilt donor plasmid library containing several types of fluorescent (EGFP, tagRFP, mCherry...selectable marker (floxed Cbr-unc-119) for easy screening of the insertion event, and a variety of connector...hundreds of genes, generating powerful genetic screening libraries. Additional protein tagging systems ...reading frame of the insertion, and 3) a vector containing the desired tag, which can be obtained as a universal...

...microhomology-mediated end-joining)? MMEJ, a form of alternative end-joining, requires only very small ...research, you know all about using non-homologous end-joining (NHEJ) to make deletions or homology-directed repair...month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction There...repair template is present. HDR also presents a cloning dilemma - the longer the homology, the more efficient...with longer homology arms comes more time spent cloning. In contrast, the short homology required by MMEJ... regions of homology, which facilitates easier cloning. An overview of PITCh. The PITCh plasmid...inserted into the PITCh vector via In-Fusion or SLIC cloning. Step 2: Design an insertion locus-specific gRNA...

...defective allele Ym and you need to convert an adenine (A) to a guanine (G)? You may wonder if a CRISPR-base editor...molecular components. Adenine base editors for plant We developed plant adenine base editor (ABE) vectors...with the desired outcome are not pictured. Adenine base editors (ABE) What if you have another gene...deaminases' which would theoretically give rise to an adenine base editor. A massive protein engineering effort...an adenosine deaminase that can act on DNA for adenine base editing. They first created a defective chloramphenicol... (H193Y). The reversion of this mutation by an adenine base editor would restore antibiotic resistance...into E. coli harboring the defective CamR gene. Screening for surviving colonies and subsequent rounds of...

...pooled libraries, from cloning a pooled library to viral production to screening. Many of Addgene’s pooled...libraries here at Addgene, including cDNA, barcoding, screening, and gRNA libraries. Most of the pooled libraries...determined, larger lentiviral preps can be used for screening-scale transduction of cells, resulting in cell...library. These cells can then be used for genetic screening, followed by sequencing and further data analysis...quality control our viral preps, we recommend determining the functional (infectious) titer once you've...as seeding density and reagent concentration.  Screening time Phew! You are now (finally!) ready to use...hesitate to email us at [email protected]. Happy screening! This blog was written by Addgenie Hannah Dotson...

... in plasmid cloning Perhaps the most widely used application of CcdB is GatewayTM cloning, where the ccdB...toxin is stable while the antitoxin is unstable, meaning that the cells must continually produce antitoxin... not inherit plasmid due to improper plasmid partitioning, it will still inherit some toxin and antitoxin...counterselectable marker in the destination vector. Successful cloning occurs when the ccdB gene is replaced by the gene...modifications in the yeast Pichia pastoris. By combining both positive and counter selection in one plasmid...plasmid, they were able to create an efficient cloning tool. The plasmid pKSCTMF contains the mazF gene...retained in less than 10% of cells while plasmids containing the plasmid addiction system were retained in...

...to select against CRISPR positive plants, but screening these can be challenging and requires specialized...requiring the destruction of plants or tissue for GUS staining or are not suited to studying large samples. RUBY...sites. These convert the amino acid tyrosine to betanin, a member of a class of pigments primarily found...order Caryophyllales, such as cacti and beets. Betanin belongs to a different chemical family than the...purple plant pigments found in all other plants, anthocyanins.   Figure 1: An adult plant expressing...selecting transformation events and am currently planning to use it to simplify my work with an Arabidopsis... to study these mutants.   Fig. 2: (A) Betacyanin is produced from tyrosine by the activity of ...

...are crisp, bright, and lacking in non-specific staining can be a challenge in the best of cases. But it... antibody, resulting in significant background staining (Figure 1A). Not all tissues will have the same...researchers can get away with species on species staining with no problem! But to be sure that your secondary...experiments. If you do find high levels of background staining by the secondary, then the following three strategies...help!     Figure 1: Species on species staining can be a problem because the secondary antibody...that you can significantly decrease background staining by mixing your primary and secondary antibodies...are a few common solutions to avoid background staining when doing species on species IHC. (A) One solution...

... would not hide behind these two fingers the ninety-nine cubits of Aristotle, nor would you mention my...where our medicines, technologies, defense, and governing policies are developed primarily by wishful thinking...culminated in the current President holding and campaigning on many views that don't line up with the scientific... us. I worried the march would be a simple, unquestioning celebration of science, at a time when scientists...Jennifer Stancill I am a research biologist by training, so of course I felt compelled to participate ...say that I was supremely proud of Nashville that morning. Despite the rain, about 4,000 supporters of science...one person vocally protesting us, he was not threatening in any way. Participating in this march proved...

...postdoctoral pay/benefits commensurate with work training and experience, better align trainee expectations...temporary and defined period of mentored advanced training to enhance the professional skills and research...Associations around Boston - From Panel Session 2: Defining the Postdoc: An Institutional Perspective. This...of the academic research workforce, and we are beginning to identify potential solutions. Keynote speaker...structural problems seem to boil down to that fact that training in the academic research environment is setup ... is this very idea that postdocs are acquiring training to become professors that is provided as an explanation... they don’t make in wages, they make up for in training. Unfortunately, these low wages also make it so...

...imaging of cellular targets Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...frankenbody (FB) construction from the complementarity determining region of an anti-FLAG scFv and a stable scFv...small epitope tag (FLAG is eight amino acids, HA is nine), multiple repeats of the tag can recruit multiple...10.1038/s41467-019-10846-1.   Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...Gabrielle Clouse While many CRISPR-based genetic screening methods have primarily used Cas9 to knock out ...the Broad Institute have expanded the genetic screening toolkit by optimizing a Cas12a-based method for.... 2021 To validate this optimized genetic screening technique, researchers identified a synthetic ...

...club of its kind, running from start to finish during the academic year and remaining affordable for all...through October 19. The BioBuilder Learning Lab The BioBuilder Learning Lab is a fully equipped, state-of-the-art...capitalize on the excitement found through experiential learning. “Students may forget the technical details they...handful of local high school teachers receiving training modules in synthetic biology has grown into a ...a project-based curriculum, BioBuilder expands learning outcomes by emphasizing the importance of critical...you can get involved with BioBuilder Details for joining BioBuilder’s programs can be found on its website...production of fire-resistant housing insulation, and cleaning up salt runoff from winter icing.BioBuilderClub...

...the tag and how many do I need? When designing a plasmid containing a polyhistidine tag, you’ll need to...single cell has tens of thousands of proteins functioning in a variety of ways. How do scientists focus...are immobilized on a bead or resin and a sample containing the protein of interest flowed through. The protein...the metal ions.      Fig. 1: A sample containing a polyhistidine tagged protein of interest (purple... (blue, green, orange) flows through the Ni2+-containing resin. The protein of interest (purple) binds...and is immobilized along with a polyhistidine containing contaminating protein (orange). A low imidazole...relatively low frequency amino acid, proteins containing two or more adjacent histidines are present in...

...Such species-on-species staining can be partially addressed with stringent staining protocols and the use...the process could make it difficult to obtain meaningful results.  Biological replicates are distinct ...antibody specificity. B, secondary antibody-only staining will allow you to see nonspecific interactions...incubated with its antigen or a small epitope-containing peptide to block the antigen-binding site. The...      Secondary antibody specificity  In indirect staining approaches, secondary antibody specificity must...inflammation. This tends to be most problematic when staining cells or tissues that are the same species as ...secondary antibodies, and labels omitted. Any signal remaining is autofluorescence and should be considered in...

...a lab we discuss the results together, explaining our reasoning to determine if the antibody is negative...cannot be completed. A 2015 review analyzing and explaining the antibody crisis states that up to 50% of ...Initiative Cell Census Network, the Wu lab will be screening over 3,000 antibodies for use in mice and human...consistent antibodies for specific markers. The Screening Criteria While choosing which antibodies to screen...uses. We therefore use both rAb and mAB in our screening. To screen the antibodies, we have developed ...to see the reproducibility and accuracy while screening. We also compare different clones of the same ...multiple people in the lab conduct “independent screening”, where they look at the same section and determine...

...Protoc. 2017 with modifications Screening pCMV-Rep78/68 Scanning Saturation Mutagenesis (SSM) Pooled...amplification protocols tend not to work as well.   Defining your library First, you’ll need to collect specific...aim for somewhere between 30x and 200x coverage, meaning that you need 30 to 200 times as many colonies ... be in direct competition with other colonies containing smaller, recombined backbones that may replicate...Re-transformation of the SPLINTR Libraries (PDF, 401 KB) Screening Zhang Lab - Multiplexed Overexpression of Regulatory...Amplification and Sequencing Protocol (DOCX, 32 KB) Screening Rinehart Lab Iterative Synthetically Phosphorylated...-Cas9 knockout and transcriptional activation screening, a protocol paper by the Zhang lab, and the Broad...

...variable amounts of experimental manipulation and screening.  A co-selection strategy for CRISPR editing in... with inefficient sgRNAs in my initial direct screening efforts, which were similar to the approach detailed...low efficiency (0.13%) and laborious handling (screening 768 F1 animals). Recovering rare editing events...conditional-lethal mutation to identify edits with minimal screening effort. Selecting for repair of a lethal mutation...to identify a single 2xFLAG knock-in by direct screening. The center images depict the power of lethal ...parental animal, are rescued progeny, which makes screening extremely rapid. This stringent selection allowed...al., 2014), inactivation of non-homologous end-joining results in a further increase in knock-in efficiency...

...Allows for selection of plasmid-containing bacteria. Multiple Cloning Site (MCS) Short segment of DNA... of cloning methods (restriction enzyme, ligation independent, Gateway, Gibson, etc). The cloning method...into. Regardless, once the cloning steps are complete, the vector containing the newly inserted gene is...an origin of replication, selection marker, and cloning site. The ease of modifying plasmids and the ability...applications. Some common plasmid types include cloning plasmids, expression plasmids, gene knock-down ...Reference includes information about molecular cloning, how to choose a plasmid vector, molecular biology...

...types of cloning methods, such as restriction enzyme cloning, Gibson assembly, or In-Fusion cloning.  NEB...Design primers for Goldengate cloning   Design primers for In-Fusion Cloning Design primers for Gibson Assembly... any cloning method. You can select from five different insert:backbone ratios. In-Fusion cloning molar...design, as well as primer design for different cloning applications. Primer3 and Primer3Plus: For general...sets for high complexity Golden Gate Reactions Cloning ligation reaction calculators These links can help...

...resistance as a marker for successful cloning. In these instances, the cloning vector typically has two separate...Posts Learn How to Use Blue White Screening to Simplify Your Cloning Experiments See Where You Can Find...cells on average! Without some means of quickly determining which cells successfully received the correct... from the bacterium's perspective – a plasmid-containing cell must replicate the plasmid in addition to...it allows a scientist to easily detect plasmid-containing bacteria when the cells are grown on selective...gene and acts as a marker for the mutation. When designing these types of experiments it is best practice... or completely removes (in the case of Gateway cloning) one cassette. Counterselection allows the scientist...