...Drosophilia Plasmid pRDB_052 (for stable fly cell lines; Hygromycin resistance gene; for genome integration... can be inserted instead, allowing everything to stay in the correct order. Most Fragmid components are...All destination vectors contain an ampicillin resistance cassette and an origin of replication, which ...ampR) Drosophilia Plasmid pRDB_051 (for stable fly cell lines; attL and attR sequences for genome...

...supernatant is collected, filtered, and titered. A major obstacle in obtaining usable lentivirus is producing a ...concentrations will need to be optimized prior to starting. We recommend calculating the ratio of the surface... adjustments, this is generally a good place to start. If you don’t anticipate that you will need large... will take longer and may increase the risk of mistakes or contamination. Finally, choice of concentration...possible. While this information provides a great starting point, it may not translate to the target cell...

...knowledge about functionality can be gained by understanding the location and transport of the protein within...localization and transport between healthy and diseased states can also provide interesting insights into disease...analysis software produces quantifiable results and statistics that can be used to compare between different...fluorescent signal) and photobleaching i.e. FP stability. One should also be aware of additional problems...of cellular complexes are important steps in understanding the function of your protein of interest in ...

...plasmids into bacterial strains, like NEB Stable. NEB Stable competent cells lack RecA, a protein that...with this AAV lysis buffer and freeze/thaw the lystate four times in a 95% ethanol and dry ice bath to... samples are quantified by comparing to an AAV standard curve, which is generated from an ITR-containing...sterile technique when handling your AAV plasmid standard and AAV transfer plasmids. It’s easy to contaminate...

...everyday What I didn’t realize was how much I was constantly negotiating, every day. As a grad student, I ... Asking questions like the below can help you understand the perspective of all persons involved and be...view the “winner” positively if they tried to understand my position and to find a creative win-win solution...I’m looking forward to. How can I negotiate my starting date with the company so they know I’m excited...respecting cultural differences is a crucial part of understanding what is important to people so that you can ...

...drug screens to identify genes that confer drug-resistance. Mutant cells are treated with a drug of interest...and gRNA distribution is analyzed in the drug-resistant population compared to a non-treated control group...enriched” correspond to genes that confer drug resistance when mutated. Findings from this type of experiment...shed light on the mechanism by which cells gain resistance to drugs and can identify future therapeutic ...population requires a massive amount of cells as starting material. Therefore, cell types that are of low...

...reducing the time it takes to image through a large z-stack (8).  One limitation of LSFMs using single side ...illumination, especially with large tissue, is that any obstacles (e.g. air bubbles or high concentration of fluorophores...neuronal labeling methods will greatly aid our understanding of the structure-function relationship of the...Jae Lee and Pantelis Tsoulfas. Jae K. Lee is an assistant Professor at the University of Miami Miller School...Cytochemistry, 59, 129-138. Pubmed. J. Huisken, and D.Y.R. Stainier. (2009). Development, 136, 1963–1975. Pubmed. ...

...author and do not represent a formal stance taken by Addgene or its staff. Additional information about gene...ecological effects. Because the consequences of such a mistake would necessarily extend far beyond the laboratory...robust to human error. Used in combination with standard barrier protocols, they can reduce the risk of...vulnerable to human error. And with gene drives, one mistake can be enough. Reversal drives are designed to ...

...find a stably folded ortholog that cleaved RNA robustly, unlike LwaCas13a, which must be stabilized by monomeric...their quest to build a robust RNA editor, the lab started with the Cas13 scaffold, testing a whopping 21 ...precise than shRNA knockdown, it still displays substantial off-target activity. By mutating residues in ...that interact with duplex RNA, they hoped to destabilize ADAR-RNA binding to decrease off-targets. Of ...

...protein standard. The density of the antibody bands can be measured against the protein standard and the...concentration may also be measured through a protein stain, such as Coomassie blue. Though more time-consuming...are separated by size on an SDS-PAGE gel. Once stained, the heavy and light chains become visible alongside...PMC2143013. Maity H, Wei A, Chen E, Haidar JN, Srivastava A, Goldstein J. Comparison of predicted extinction...

... Albert Einstein might have put it best when he stated, “the value of an education . . . is not the learning...Provost, then a new faculty member at Minnesota State (and now Professor of Chemistry and Biochemistry... enzyme he studied during graduate school. As a stable, easily purified protein with easily measured activity...Professor and Chair of the Biology Department at Keene State College in New Hampshire. He mentors undergraduates...Scientific Elite: Nobel Laureates in the United States. Free Press. New York. Resources MDH CUREs Community...

... efficiency to read more about optimizing this distance. Homology arms and types of donors To make sure...cells are actively cycling through the cell cycle stages. If cells are overly confluent or don’t have sufficient...responsible for the transition out of S phase, so cells stay in an HDR-promoted cycle longer. This approach may... edits of small insertions and deletions.   Start your knock-in experiment today! Get ready to identify...18): 6461 (2020). 10.3390/ijms21186461 Lin, S., Staahl, B. T., Alla, R. K., Doudna, J. A. Enhanced homology-directed...

... could infect non-dividing cells, but without sustained transgene expression. To design a viral vector...Lentiviral vectors are also commonly used to make stable transgene-expressing or knockout cell lines. Many...research is still in the pre-clinical/early clinical stages. Researchers are also working to make lentiviral... nearby genes. These safer vectors have become standard in gene therapy. Researchers have also developed...below! References 1. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral...

...HNH endonuclease domain. Cpf1 cleaves DNA in a staggered pattern and requires only one RNA rather than ...cleavage and G-rich PAM requirement. Zetsche et al. started from square one to characterize the Cpf1 nucleases...nt upstream of the PAM site, Cpf1 cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18...amino acids long respectively), both cleave in a staggered pattern similar to FnCpf1. Potential advantages... another option to the CRISPR toolbox. Cpf1’s staggered cleavage pattern opens up the possibility of directional...

...size. From the pool of shorter orthologs, only Staphylococcus aureus Cas9 (SaCas9, 1053 amino acids) displayed...genome modification, but they did so without a substantial immune response or toxicity. More AAV-based CRISPR...Harrington LB, Al-Shayeb B, Wagner A, Brötzmann J, Staahl BT, Taylor KL, Desmarais J, Nogales E, Doudna JA...Dohmae N, Ishitani R, Zhang F, Nureki O (2014) Crystal Structure of Cas9 in Complex with Guide RNA and...Zhang F (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186–191 . https://doi.org...

... time-consuming, and complex to use outside of standard laboratories. In the case of the COVID-19 pandemic...SHERLOCK to detect SARS-CoV-2 RNA. The test is started using RNA purified from patient samples and can... a different PAM site and generates 5’ and 3’ staggered ends after dsDNA breaks. The Doudna lab discovered...applications. The company Mammoth Biosciences has been started based on DETECTR technology with the mission to...s41587-020-0513-4 Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA (2018) CRISPR-...

...destination vector carry different antibiotic resistance markers (indicated here by plasmid color), allowing...How to clone using Gateway technology To better understand the process, we’ll walk through an example experiment...expression elements (ribosome recognition sequences, start codon, stop codons, reading frame considerations...Gateway cloning kit, and MultiSite Gateway plasmids. Constant reading frame When you move a DNA fragment from...Gateway vector to another, the inserted DNA fragment stays in frame. High efficiency The positive (antibiotic...

...these different transcriptional start sites in order to better understand isoform-specific gene regulation...each gene isoform has a distinct transcriptional start site. Because CRISPRa can be targeted anywhere in... Bdnf gene possesses 9 distinct transcriptional start sites, all of which yield the same BDNF protein....downstream of the TUJ1 coding sequence. ES cells that stably express the dCas9-Suntag activator were transduced...

...controls for flaws. Recently the Bassik Lab at Stanford developed a new genome-wide CRISPR knockout screen...via a lentiviral vector to cell lines that either stably expressed Cas9 or that had been lentiviral infected...also lead to fewer false-positives results (i.e. mistakenly calling a gene a hit when really it’s not). In...the PAM are less tolerated than mismatches more distal to the PAM, and 2) guides with high GC content ...error rates in genomic perturbation screens: gold standards for human functional genomics." Molecular systems...

...protein is up or downregulated, examine a protein’s stability or post-translational modifications, or study ...A and Protein G, which bind to the fragment crystallizable region (Fc) of antibodies with a high affinity...conformation. Frequently, the antibodies used for a standard, denaturing Western blot will not be suitable ...interest, such as a sample transiently transfected or stably infected with the protein of interest and a sample..., Hjelm K, Sjöquist J (1972) Protein a from Staphylococcus aureus. Its isolation by affinity chromatography...