...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 μg of DNA μL of 1 mg/mL PEI 1:...Centrifuge the viral supernatant at 2100 rcf for 5 minutes to pellet any packaging cells that were collected...

...Biosafety Cabinet 4 °C Refrigerator Micropipettes and tips Pipettor and pipettes Benchtop centrifuge compatible...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...Reagent Preparation instructions. Wipe down all pipettes and equipment with 10% bleach prior to use. Reagent...

...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...

...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...

...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...

...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...

... 0.5-1μL T4 DNA Ligase H 2 O to a total of 10μL Notes: If the DNA concentrations are low such that you...troubleshooting failed ligations. The following table indicates the various controls: Control Ligase Interpretation...

...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment... Equipment Pipette controller Pipette tips and pipettes Rocking platform Tweezers Fluorescent microscope...

...lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to find one ...solution through a 0.22 μm filter to remove any precipitates. Stain each well with 1 mL of 0.1% crystal violet...

...also needs to avoid primer-primer annealing which creates primer dimers and disrupts the amplification process...

...2 mL screw top tube or cryovial and gently mix. Notes: Make the 50% glycerol solution by diluting 100%...