... 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... permeabilizing cells Gently aspirate the media from the 24-well plate. Wash each well with 500 µL of ...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...Sterile Poly-D-lysine coated coverslips HeLa cells 24-well plate 4% Paraformaldehyde 5 mg/mL 4′,6-diamidino... the microscope slide with the cell side facing down. Observe the cell labeling on a microscope with appropriate...

...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...

... 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...

... minutes) to pellet bacteria. Decant LB and weigh pellet. The total weight of each pellet should be ~1...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...

...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...

...a mouse. Procedure Section 1: Lyse cells Centrifuge 5 x 10 6 cells for 5 min at 100 x g . Gently remove...remove supernatant. Resuspend cell pellet in 1 mL of 1X PBS and transfer to a microcentrifuge tube. Centrifuge... g . Carefully remove supernatant. Resuspend cell pellet in appropriate volume of cold lysis buffer. Pro-Tips...buffer will vary depending on the size of your cell pellet but will generally be between 250–1000 µL . The...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...

... Wash the pellet or column with 70% ethanol to remove excess salt. Resuspend the DNA pellet, or elute ...careful not to disturb the bacterial pellet. Resuspend the pellet in 100 μL of cold Solution I. Vortex... tube for 5 min at 12,000 g. Notes: Pellet contains proteins, cell fragments, salt and other extra particles...Optional Wash the pellet with 70% ethanol. Note: This step removes excess salt from the pellet which can cause...the sink. Pro-Tip Be careful, the pellet is harder to see and less well attached to the tube after the 70%...Invitrogen , and (Link opens in a new window) Promega sell kits for isolating plasmid DNA in quantities as ...require larger cultures). Centrifuge the culture to pellet the bacteria before proceeding with DNA preparation...

...Axygen, PCR-02-FCP-C ddPCR 96-well PCR plates, Bio-Rad, 12001925 48-well dilution plate, Bio-Rad, MLL4801...the 96-well plate sealer by gently touching the screen. Prepare the Serial Dilution Place a 48-well dilution...10 sec before use. Place an 8-well PCR tube strip into a chilled 96-well freezer block. Add 20 µL of the...touch the bottom of the well. Lift the tips ~1 mm. Touch the side of the well and tilt the pipette tips...Centrifuge, Thermo Scientific, 10199-452 Ice bucket 96-well freezer blocks (x 3) Reagents Molecular Biology ...dilution plate in a chilled 96-well freezer block and place in the dilution BSC. Prepare 1X dilution buffer... µL of each viral sample to Dilution 1 in the 48-well dilution plate and pipette 5–10 times to mix. Dilute...

...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...

...above the well. Very slowly and steadily, push the sample out and watch as the sample fills the well. After...perform gel electrophoresis. Equipment Casting tray Well combs Voltage source Gel box UV light source Microwave...the gel. Pour the agarose into a gel tray with the well comb in place. Pro-Tip Pour slowly to avoid bubbles...the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with ...sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer. Once solidified...added EtBr to your gel, add some to the buffer as well. EtBr is positively charged and will run the opposite...of the gel. Note: When loading the sample in the well, maintain positive pressure on the sample to prevent...