...Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells Day 7: Harvest antibody Equipment...10 µL of the cell suspension/trypan blue mix into a cell counting chamber. Load the cell counting chamber...interest in cell culture. This protocol describes how to transfect suspension HEK293 cells with recombinant...Section 1: Seeding cells The day prior to transfecting, seed a 108 mL culture of HEK293 cells at a density ...Pro-Tip Do not use cells that are over 30 passages. Section 2: Transfection Check the cell density and viability...transfer 0.5 mL of HEK293 cell suspension into a clean microcentrifuge tube. Pro-Tip Cells settle quickly and...microcentrifuge tube. Vortex the cell suspension. Transfer 10 µL of cell suspension into the microfuge tube...

... of HEK293T cells optimized for viral production (AAVpro or Lenti-X), but any HEK293T cell line should...different cell lines and different transfection reagents. Workflow Timeline Day 0: Seed HEK293T cells (or a... health of the cell line is critical for obtaining high levels of virus. HEK293T cells should be split...Plate 1x10 6 cells in a 75 cm 2 flask in a volume of 15 mL. Wednesday: Plate 1x10 6 cells in a T75 flask...8x10 5 cells in a T75 flask in a volume of 15 mL. Do not add antibiotics to the media. Use cells that are...PEI determined. Procedure Seed HEK293T cells at 3.8x10 6 cells per plate in DMEM complete in 10 cm tissue...prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1–2 days after...

...such as stable-cell line generation. Workflow Timeline Day 0: Seed 293T packaging cells Day 1 (pm): Transfect...and for each cell line. Considerations Before You Start The health of the packaging cell line is critical... viral titer. 293T cells should be split 3 times a week: Monday: Plate 1×10 6 cells in a T75 flask in ...: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM Complete. Friday: Plate 8×10 5 cells in a T75 flask .... Use cells that are below passage 15 for viral production. Procedure Seed 293T packaging cells at 3.8....8×10 6 cells per plate in DMEM Complete in 10 cm tissue culture plates. Incubate the cells at 37 °C, ...prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 days after...

...commonly referred to as 'competent cells.' Many companies sell competent cells, which come frozen and are prepared...that came with your competent cells. Pro-Tips Commercial competent cells range significantly in their ...SOC media Competent cells DNA you'd like to transform Procedure Take competent cells out of -80°C and thaw...use electro-competent cells. Instead of relying on the heat-shock to cause the cells to take up the DNA,...process by which foreign DNA is introduced into a cell. Transformation of bacteria with plasmids is important... all plasmids (even those designed for mammalian cell expression) carry both a bacterial origin of replication...transformation efficiency. The lowest efficiency cells (usually the least expensive) are fine for transforming...

...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...

...1: Seed and transduce cells Day 4: Treat cells with Benzonase and harvest cells Day 4+: ddPCR and analysis...viruses per cellular genome: Since the cellular gene RPP30 is used as a reference to the cellular genome, ... target cells but can be modified for other targets. Primers and a probe against the cellular ribonuclease...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...

...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...

... 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...

...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...

...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 ...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...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...

...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...

...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... 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...

...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...

... 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...

...how to affinity purify recombinant antibodies from cell culture supernatant using Protein A or Protein G...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...after use. Carefully pour the filtered and diluted cell culture supernatant into the Gravitrap column. Collect...

...bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria... and you will not need to obtain more competent cells and retransform. Bacteria on an LB agar plate can...

...like microbes in a drop of pond water or the cells in your cell culture dish. Microscopes come in a huge ...transmission electron microscopes that allow us to see cellular components with science-fiction levels of detail...

...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...samples may not have been processed correctly in the cell (e.g., failure to cleave the signal peptide) and...Scatter chart. Change the data range and select the cells where the x-axis is represented. Add a linear trendline...

...complete plasmid can be transformed into bacterial cells for propagation. The majority of ligation reactions...Interpretation Uncut vector - Checks viability of competent cells and verifies the antibiotic resistance of the plasmid...