...detect antigens in cells. Here we describe the basic steps for fixing and labeling cells in culture with ...2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells Equipment Pipette controller Pipette... Seeding cells Place a sterile poly-D-lysine coated coverslip in each well of a 24-well cell culture treated...treated plate. Seed 5 x 10 3 HeLa cells per well. Allow the HeLa cells to grow to the desired density before... the microscope slide with the cell side facing down. Observe the cell labeling on a microscope with appropriate...describes the basic steps for fixing and staining cells in culture with a primary antibody against a target...outlines the steps for fixing and labeling HeLa cells for a target protein using the formaldehyde fixation...

...limited. Day 2: Harvest cells and purify DNA (Estimated time 3-4 hours) Cells should be harvested first...electrocompetent cells (Default: 4 tubes of Endura Duos, Lucigen, 60242-1) Alternatives include Stbl4 cells or other...Maxipreps rely on incremental, ordered cell lysis. E. coli cells are subject to lysis by freeze-thaw if...Ensure that electrocompetent cells are being used. Chemically competent cells will not provide adequate ...CRISPR pooled plasmid libraries in Escherichia coli cells. Protocols...of a pooled-plasmid library in Escherichia coli cells. Pooled libraries contain tens to millions of different...other ultra-high efficiency electrocompetent cells that are suitable for unstable or recombination-prone...

...Generating Stable Cell Lines with Lentivirus Genomically integrate your DNA sequence into a cell line Isolating...Isolating a Monoclonal Cell Population by Limiting Dilution Generate monoclonal cell lines from a polyclonal...polyclonal pool of stable cells AAV Production in HEK293 Cells Produce adeno-associated virus to deliver your...Extraction Without a Kit Extract RNA from tissues or cells without a commercial kit Plasmid Cloning Name Description...Transfection Introduce plasmid DNA to mammalian cells Lentivirus Production Produce lentivirus with a ... virus from a preparation produced in mammalian cells Watch the Video! AAV Titration by qPCR Using SYBR...Transfection Introduce plasmid DNA to mammalian cells to produce antibodies Antibody Purification Purify...

...1: Seed and transduce cells Day 4: Treat cells with Benzonase and harvest cells Day 4+: ddPCR and analysis...wells with 3,000,000 cells in 13.5 mL of media and 11.1 µg/mL Polybrene. Mix the cell suspension well before...each well. Detach cells by incubating with 200 µL TrypLE for 1–2 min. Resuspend cells in 500 µL DMEM complete...above, the cells per well is 300,000 and the virus volume refers to the total volume the cells were transduced...used to transduce HEK293T cells. Genomic DNA is extracted from the target cells and assayed for integrated... target cells but can be modified for other targets. Primers and a probe against the cellular ribonuclease... (cations can affect the attachment of adherent cells) Trypsin EDTA (TrypLE, Thermo Fisher, 12605010) ...

... or cells without a kit. The general steps of this protocol include homogenization/lysis of cells or tissues...lyse tissues or cells in Solution D. For tissues: use 1 mL of Solution D per 100 mg of cells. For cultured...cultured cells: use 1 mL of Solution D per 1 X 10 7 cells. Allow sample(s) to sit at room temperature for 5...effectively lyse open the cells. Extract RNA from the homogenized sample(s).Transfer tissue/cell lysate to a 4 mL...tissues or cells in TRIzol® or a similar product. For tissues: use 1 mL of TRIzol® per 100 mg of cells. For ...For cultured cells: use 1 mL of TRIzol® per 1 X 10 7 cells. Allow sample(s) to sit at room temperature for... RNA isolation will depend on how effective your cell lysis protocol is. While simple homogenization is...

...a mouse. Procedure Section 1: Lyse cells Centrifuge 5 x 10 6 cells for 5 min at 100 x g . Gently remove...This protocol describes the basic steps for lysing cells, determining total protein concentration in the ...use western blotting to visualize a protein from cells or tissue samples. Equipment Microcentrifuge 0.5...remove supernatant. Resuspend cell pellet in 1 mL of 1X PBS and transfer to a microcentrifuge tube. Centrifuge...100 x g . Carefully remove supernatant. Resuspend cell pellet in appropriate volume of cold lysis buffer...lysis buffer will vary depending on the size of your cell pellet but will generally be between 250–1000 µL...

...investigator to conveniently plate and select transformed cells containing plasmids differing in their resistance...bent to create an “L” shape, and then used like a cell spreader. Several other devices may be used for.... During the incubation, transform DH5α E. coli cells by heatshock with the plasmid of interest. See our...the liquid is mostly absorbed. The spreading of cells can be done in the same way as the antibiotic, using... using either a bent micropipette tip or other cell spreading device that fits the plate. Incubate plates...

...use electro-competent cells instead of the more common chemically-competent cells. The number of bacterial...might need to express YGOI in cultured mammalian cells. The problem is that the only version of full-length... manufacturer’s instructions for your competent cells. For most standard cloning, you can transform 1-...1-2μl of your ligation reaction into competent cells such as DH5alpha or TOP10. If using much less total...colonies, you might want to use higher competency cells. Additionally, if your final product is going to...

... CRISPR Generate genomic deletions in mammalian cells lines using CRISPR/Cas9 Genomic Deletions with CRISPR... virus from a preparation produced in mammalian cells AAV Purification by Iodixanol Gradient Ultracentrifugation... the lab. Blog post: 10 Basic tips for mammalian cell culture Aseptic Technique Aseptic technique is a...fields such as microbiology, molecular biology or cell biology. Learn some of the key strategies of aseptic...

...bacterial cell. Large plasmids usually have a low copy number (approximately one or two copies per cell) and... can be present in large numbers, 50 or more per cell and have a high copy number. High copy number plasmids...increase this to 350 - 400 rpm to obtain a higher cell density. Reference Page Top Index...