...sample. The isotype antibody will be virtually identical to the antibody for your marker of interest. It...research technician in Arlene Sharpe's lab at Harvard Medical School.   More resources on the Addgene blog Introduction...

...anti-FLAG-FB-GFP binding to FLAG-tag H2B for nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling...4xFLAG-mCherry-β-actin), and mitochondrial (mito-mCherry-1xFLAG) localization. Adapted from Liu et al. 2021. Anti-FLAG...

...end joining (NHEJ). In these reporters, editing typically results in a change in the fluorescent readout...stimulated homology-directed repair. Journal of Biological Chemistry 292:6148–6162 . https://doi.org/10.1074...

... specific DNA base changes and consist of a catalytically impaired Cas protein (dCas or Cas nickase) fused...previously uneditable sequences. Four DNA base swaps, so called point mutations that change C to T, G to A, A to...

...powered plasmid maps far easier to navigate. These practical and effective changes will give you the ability... and “Sequence” tabs. BLAST Use the Basic Local Alignment Search Tool (BLAST) via the NCBI website...

... the following, which are later printed for my physical lab notebook, along with any figures generated...between each word can make it difficult to accurately call a file. Version control your commonly used scripts...

...once it is engaged by its target, it will non-specifically cut ssDNA. This feature is shared by other Cas... potential as a viral therapeutic that could specifically degrade target viral RNA. For more information...

...out the sources of miscommunication and b) find practical solutions.  My PhD prepared me remarkably well...answer to these questions--which were real, not hypothetical--was clearly “no.” When there was room for misinterpretation...

...intuitive, or logical to a biologist that are not at all obvious, intuitive, or logical to a layperson...

...library, propagated it, separated it into roughly identical replicate groups, and treated these groups with...would have different labels - i.e. they would stochastically arise from different cells throughout the treatment...

...Another strength of Golden Gate cloning is its scalability. Unique 4 base overhangs can be used to assemble...design of flanking overhangs. Although there are theoretically 256 distinct flanking sequences, sequences that...

... will provide an initial consultation meeting or call for free.  Take advantage of this to get the information...blogged for us) offers a free report on her website called “Secrets to Success in Graduate School”. She has...

... for PAM-less editing   Guides for CRISPR are typically selected by the ‘quality’ of the gRNA sequence...the Liu lab through phage-assisted evolution, specifically with the goal of generating Cas enzymes with...

...search algorithm rather than pre-designed sets, so identical searches can return multiple answers. You might...might want to take advantage of their save and recall functions when you get results you like. NEBridge ...

...given the data provided by the depositing lab, we call these “quality control (QC) issues.” We then ask..., Brigham MD, Sanjana NE, Zhang F (2017) Genome-scale CRISPR-Cas9 knockout and transcriptional activation...

...to visualize DNA, an editor targets DNA (via catalytically inactive dCas9) to modify chromatin. A fluorescent...INTRSECT project. Check out the new additions.  Image calcium transients in PV interneurons with AAV-S5E2-GCaMP6f...

... Roska’s lab at the Institute of Molecular and Clinical Ophthalmology Basel recently took a different ...Strategy #2 (ProB): Create promoters using phylogenetically conserved sequences that are upstream of the...

... gRNA (Anzalone et al., 2019), disrupting its canonical structure and Cas9 binding (Nishimasu et al., ...other bases in this location bind to Cas9 with canonical sgRNA structure (right). Created with BioRender.com...

...characters. A descriptive purpose focuses on the practical application of the plasmid and will help a scientist... Lysine (D123K), lowers biosensor affinity for calcium.”  Primary Vector Type The vector type should...

...Note: Cpf1 is also called Cas12a. In 2015, Feng Zhang’s lab characterized two Cpf1 nucleases, distant...should remove the PAM sequence. Many research and clinical applications require HDR over NHEJ, so comparing...

...interest, and the two helper plasmids, commonly called “triple plasmid transfection” (Figure 1).    Figure... ddPCR- and qPCR-based titering assays measure physical titers, meaning the concentration of viral particles...

...efforts make a precise edit or insertion via a chemical modifier. We used these categories to see what...function from 2012 to 2023; data are smoothed using locally estimated scatterplot smoothing. (C) Number of ...

...not as quantitative as other methods of PCR. Theoretically scientists should be able to determine the quantity...addition of a probe based fluorescent dye that intercalates with any dsDNA and the use of a fluorometer ...

...however, your cost per cloning reaction drops dramatically, as one tube of enzyme should be enough for ... guarantees, some cloning techniques do tip the scales in your favor. For starters, Golden Gate cloning...

...into the genome, CRISPR may increase this rate. Specifically, portions of the AAV genome can integrate at...AAV episome-sequencing (CrAAVe-seq) is a highly scalable cell type-specificin vivoscreening platform. bioRxiv...

...contracts behind closed doors. Fig. 1: The mythical land of negotiations.    You negotiate everyday...are negotiating for a specific time slot on the confocal microscope. You’re trying to get better images...

...sequence (gene). In a clean sample, there are typically fewer than 10 hits to a given sequence, and we...control is recommended but not yet required for clinical-grade materials, and is not commonly performed...

...directions. Drosophila are very easy to manipulate genetically and have limited genetic redundancy (meaning,...reference of various fly phenotypes, check out the Classical Genetics Simulator website. One interesting thing...

...requires incubation at 55 °C - a dangerous, non-physiological temperature for mammalian cells and one which...results out there include: Google Groups - I've specifically set up a forum where NgAgo users can share protocols...

... to deaminate cytidine and used dRanCas13b (catalytically inactive Cas13) to target the cytosine deaminase... from these off-target edits helped to minimize local off-target editing by RESCUE. To address both C-...

...quickly along regions calling for abundant, charged tRNAs but will stall at regions calling for low abundance...

...but essentially we just needed to send a Google calendar invite over to the r/Science team with a description...recommend taking every opportunity you get to speak publically about your work (you can find my tips for a perfect...

...guest blogger Samantha Young. Samantha Young is a Medical Writer with a PhD in reproductive biology and genetics...Mashiko, Daisuke, et al. "Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid...

...the Qiagen HiSpeed Maxi Kit (one conical is its own Maxiprep). Critical Do not freeze pellets for later...libraries up to 200,000 individual plasmids and can be scaled according to the actual number. Required Quality...selection). This recombination, at a low rate, is not typically a problem as these sequences are not efficiently...200,000 individual plasmids. For larger libraries scale the number of cells and DNA accordingly. Pro-Tip...cells) Less library DNA can be used but this dramatically increases the chances of individual plasmids...Cell Spreaders (VWR, 60828-680) 5 mL and 10 mL Serological pipettes Ice slurry (Ice bucket with ice and ...normal growth is expected to work. 50 mL Falcon Conical tubes (Fisher, 14-432-22) Reagent Preparation Prepare...

...that affect the frequency of multimerization. Theoretically, as long as all the plasmid features remain ...PMCID: PMC225270. Higgins NP, Vologodskii AV. Topological Behavior of Plasmid DNA. Microbiol Spectr. 2015...

... forget to factor this into your calculations later (see calculation example below). Seed 300,000 cells... a CSV file which will be used to calculate the titer. Calculations In the experimental setup above, the...20 200 40 400 80 800 160 1600 To calculate the titers, first calculate the number of viruses per cellular...) to titer lentivirus vectors. This protocol specifically uses primers and probes targeting integrated...ddPCR and analysis Equipment Class II, Type A2 Biological Safety Cabinet Aspirating unit Microcentrifuge...glutaGRO, Corning 25-015-CI) 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...cartridge holder with bleach and place it in the Biological Safety Cabinet (BSC). Make sure that the BSC ...

...the full plasmid sequence can be assembled or theoretical — it does not have to be entirely verified by... Lysine (D123K), lowers biosensor affinity for calcium". Please describe any tags or fusions (e.g. His...

...Complete to a 15 mL conical and an appropriate amount of 25 mM chloroquine to the conical so that the final...am): Harvest virus Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette... Da Heat-inactivated FBS 1x PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...10 cm tissue culture dish, Corning 430167 15 mL conical tubes, VWR 21008-216 Hydrochloric acid Sodium hydroxide... or sodium hydroxide to adjust the pH to 7.0. Typically, the solution will be basic and will need adjustment...optimal mass DNA:mass PEI ratio will need to be empirically determined for each new batch of 1 mg/mL PEI ...Start The health of the packaging cell line is critical for obtaining high viral titer. 293T cells should...

... of PBS. Calculate the fraction of fluorescent-positive cells in each well. When calculating titer, only...fluorescent cells Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...mL), Millipore TR-1003-G 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008-216 Lentivirus preparation Reagent...You Start The health of the target cell line is critical for obtaining accurate titers. Check the cells...remaining well, a cell count is required for calculating the titer. Incubate for 48–72 h. Gently aspirate...leading to underestimation of the true titer. Calculate the transduction units per mL (TU/mL) using either...

...Nuclease-free water Microcentrifuge tubes 96-well optical plate Pipette tips General Considerations Always...should also contain an internal passive reference (typically ROX dye), to normalize non-PCR–related fluorescence...Addgene. The values highlighted below in red were calculated using this plasmid, but will change if you use...use a different plasmid. Sample Calculation Size in bp of Addgene plasmid #59462 : 6208 bp Concentration...vortex) Incubate 30 min at 37 °C Transfer to ice ** Critical: do NOT treat your plasmid standard with DNase..., use dilutions 5–8 Note: at Addgene, samples typically range from 1 x 10 12 GC/mL to >2 x 10 13 GC/mL...Pro-Tips The quality of the sample dilution series is critical. Make sure to pipet each dilution up and down ...