We narrowed to 1,006 results for: CAL

...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 ...

...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-...

... 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 ...

...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...

...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...

...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...

... 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...

...for short term studies of protein function. We specifically use this protocol with a subclone of HEK293T...perform your experiment Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette... or sodium hydroxide to adjust the pH to 7.0. Typically, the solution will be basic and will need adjustment...Before You Start The health of the cell line is critical for obtaining high levels of virus. HEK293T cells...optimal mass DNA:mass PEI ratio will need to be empirically determined for each new batch of 1 mg/mL PEI ...batches of PEI depending on the user, quantities of chemical used, volumes, pH adjustment etc. Consequently...Pro-Tip The ratio of µg DNA:µg PEI needs to be empirically determined. Once a batch of PEI is prepared, ...

...backbone of the two DNA fragments. This reaction, called ligation, is performed by the T4 DNA ligase enzyme...nucleotides together. After ligation, the insert DNA is physically attached to the backbone and the complete plasmid...digestion . Most restriction enzymes digest DNA asymmetrically across their recognition sequence, which results...digested end of the DNA fragment. The overhangs, called "sticky ends", are what allow the vector and insert.... Use a (Link opens in a new window) ligation calculator to easily quantify how much vector and insert...of DNA, buffer and ligase into a 10μL reaction, scale the reaction size as necessary - being sure to increase...vector end. (Link opens in a new window) Ligation calculators are easily found on the web. Just enter the concentration...

...flasks). Cell stacks provide an efficient means to scale-up without having to handle a large number of T-...am): Harvest cells Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette...Thermo Fisher, 12605010) 1X PBS pH 7.4 without calcium or magnesium, Corning 21-040-CV (cations can affect...Before You Start The health of the HEK293T cells is critical for optimal AAV yield. Do not overgrow your cells...incubator for 24 h–36 h. Proceed with transfection: Calculate the amount of each plasmid needed to have a 1:... Therefore, for each plasmid we need: Sample Calculations RepCap: 0.08 μg/bp × 7,265 bp = 582.1 μg Volume...detach easily. Transfer cells and media into 500 mL conical vessels. Rinse CS5 once with 100 mL of PBS and ...

...gene. For example, the Whitehead Institute for Biomedical Research hosts an siRNA Selection Program that... #A9414 Luria Broth Agar (LB agar) American Bioanalytical: #AB01200-02000 Ampicillin VWR: #7177-48-2. ...transfection in a 6 cm plate. The protocol can be scaled to produce different amounts of virus as needed... transfection reagent in serum-free OPTI-MEM. Calculate the amount of Fugene® and OPTI-MEM necessary given... Sigma-Aldrich: #H9268 Protamine Sulfate* MP Biomedicals: #194729 Puromycin* Sigma-Aldrich: #P8833 Target...0.5 mL for a high MOI, and 0.1 mL for a low MOI). Scale the amount of virus added depending on the size ...antibiotic Per 40 grams of powder from American Bioanalytical catalog # AB01200-02000, LB contains: 10g tryptone...

...resistant colonies 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...required to kill your target cell line needs to be empirically determined. Treat the target cells with a range... can usually be omitted because this dilution typically produces a lawn of cells, which cannot be accurately...integration events decreases as the dilution increases. Calculate the transduction units per mL (TU/mL) as follows...

...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...

...analyzing the gene’s function. The process is shown graphically in the following cartoon, in which we are adding...TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a final Reverse Primer sequence of 5’-...reaction and 1μg of recipient plasmid. It is also critical that as much of the recipient plasmid as possible...step, depending on the phosphatase you choose. CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase...base pairs for each varies, it is difficult to calculate this based on DNA concentration alone. One method...electro-competent cells instead of the more common chemically-competent cells. The number of bacterial colonies...will tell you your “background” level or more specifically it will tell you how many colonies you can expect...