...doesn’t affect any endogenous restriction enzyme sites? Never fear! You can introduce a silent, passenger...CRISPRitz or CRISPOR can help narrow down the list of sites where off target editing may occur, allowing you...

...misfolding that leads to a build-up of protein aggregates termed “lewy bodies” in the brain. Yeast models...majority of C. elegans are also self-fertile hermaphrodites making it easy to conduct large experiments...

... for the majority of documented phosphorylation sites. If knowledge of a candidate regulatory kinase is...that binds specifically to phosphate moieties and promotes a robust mobility shift in phosphorylated, but...

...transiently-transfected cells, increases reproducibility, as it eliminates the variation associated with repeated transient...and FBS lots can promote or inhibit transfection. Test a variety of brands and FBS lot numbers to find ... expanded, prepare cell stocks and/or harvest to test for protein expression. Typically, cells transduced...levels of the transgene may have reduced growth rates, especially if that transgene is toxic. Eventually...

...secondary antibody only labels sites where you expect the target to be. You can test this by preparing a sample...

...primers, and probe on ice before use. Wipe down all pipettes and surfaces with 10% bleach. Safety Warnings ...PCR tubes, Axygen, PCR-02-FCP-C ddPCR 96-well PCR plates, Bio-Rad, 12001925 48-well dilution plate, Bio-... 1X PCR buffer (1:25,600,000) Use multichannel pipettes for the dilution series. For dilutions 1–5, use...reduce NTC values, we recommend wiping down all pipettes and equipment with 10% bleach prior to use and...

...lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to find one ...inactivated in the lab by heating to 56 °C for 30 minutes. 0.45 μm polyethersulfone (PES) filter system, ...and add 35 mL of 0.05% Trypsin/EDTA. Wait ~2-3 minutes for cells to detach. Gently tap the sides of the... and sonicate 5 x 1 sec pulses with at least 5 minutes on ice between each pulse, 50% amplitude. Return...

...). In vivo protein labeling with trimethoprim conjugates: A flexible chemical tag. Nature Methods, 2(4..., G., Tünnermann, J., Tänzer, T., Karpenko, J., Mertes, N., Xue, L., Uhrig, U., Reinstein, J., Hiblot,...

...mixes and samples on ice before use. Wipe down all pipettes and surfaces with 10% bleach. Safety Warnings ...PCR tubes, Axygen, PCR-02-FCP-C ddPCR 96-well PCR plates, Bio-Rad, 12001925 Pierceable foil heat seal, Bio-Rad...Cell Harvest Gently aspirate media from the 6-well plates. Be sure to use a new aspirating pipette for each...and Troubleshooting We recommend wiping down all pipettes and equipment with 10% bleach prior to use and...

...with standard agarose gel electrophoresis, which separates DNA by their length in base pairs. Following electrophoresis...Electrophoresis Protocol with the following amendments: Notes: Gel purification is most efficient with lower %...blade, slice the desired DNA fragment from the gel. Notes: To protect the UV box, it is a good idea to place...

...commonly used to culture bacteria in the lab. LB agar plates are frequently used to isolate individual (clonal...characterized by a cloudy haze in the media (see right). Notes: Some protocols require bacteria to be in the log...on your plasmid. If the bacteria on your LB agar plates are not fresh, you should streak your bacteria ...

...restriction sites. See (Link opens in a new window) NEB's table of methylation sensitive restriction sites . Sometimes...similar, but not identical, to their recognition sites. This is due to "Star Activity" and can happen ...

...lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to find one ... plate in DMEM complete in 10 cm tissue culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h...the table below for a possible range of ratios to test: Ratio of DNA:PEI Amount of DNA (μg) Volume of 1...

...Gibson Cloning? No need for specific restriction sites. Join almost any two fragments regardless of sequence...unique function for the reaction: T5 Exonuclease - creates single-strand DNA 3’ overhangs by chewing back ...other. Phusion High-Fidelity DNA Polymerase - incorporates nucleotides to “fill in” the gaps in the annealed...

...ultracentrifugation. The arrow indicates the 60–40% interface. The vertical black line indicates the location of the...fractions once you have a good idea when the virus elutes from the gradients. In a biosafety cabinet, carefully...

...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...tubes, VWR 76289-498 Before Starting Wipe down all pipettes and equipment with 10% bleach prior to use. Warm...Spectrophotometer using the A280 IgG setting. Combine all eluates with measurable protein. Determine the protein ...

...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...January 24, 2022 Workflow Timeline Day 1: Prepare lysates, run SDS-PAGE, transfer, block, incubate with primary...channel pipette Pipette controller Pipette tips and pipettes Spectrophotometer Heat block Mini gel tank chamber...

...experiment. Read More Optogenetics Optogenetics integrates optics and genetic engineering to measure and...

...maxiprep). If delivering as virus, make virus; this creates a pooled lentiviral CRISPR library. Apply pooled...

...on the surfaces of the water bath as the water evaporates from the tub. Disinfectant may be added to prevent...heat up quicker. Pro-Tip Set up the water bath 30 minutes to 1 hour before you need to use it to allow the...