...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...

...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...

...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...

...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 ...

...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 ...

...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...

...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-...

...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...

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

...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 ...

...lines of interest Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...CI) Heat-inactivated FBS 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...selective medium is used, it may have additional biological effects on the cell line. In the absence of selective... medium throughout experiments be determined empirically, and that the cell population be monitored regularly...individual cells by passing several times through a serological pipet or by passing through a 40 µm cell strainer...will be used to seed the 96-well plate. Sample Calculation First, use a hemocytometer to quantitate the ...well plate, make 10 mL of a 5 cell/mL solution. Calculate the total cells needed: Total cells needed: 10...