...into each well of a 96-well plate. By doing this, you are seeding the plate at an average density of 0.5...scan the plate for individual cells, which should be on the same focal plane. Scan the entire plate and make...the 96-well plate, use a microscope to ensure that the cells have detached from the plate and can be isolated...days. Single cells were then plated in individual wells of a 96-well plate and left undisturbed for 13 ...details) Day 1: Seed individual cells in a 96-well plate Day 2–14: Monitor cells for growth and expand cells...tissue culture treated dish, Corning 3516 96-well plate, Corning 3596 0.45 μm polyethersulfone filter, Nalgene... and the specific conditions used. Each 96-well plate described in this protocol could, in theory, give...

...02-FCP-C ddPCR 96-well PCR plates, Bio-Rad, 12001925 48-well dilution plate, Bio-Rad, MLL4801 Pierceable... Foil Heat Seal on the PCR plate with the red line facing up. If the plate sealer is not at temperature...place the PCR plate with the foil onto the metal support block. Place the block in the plate sealer and ...transfer the plate to the Droplet Reader. Open the QuantaSoft software to set up a new plate layout. Designate...Bio-Rad, DX200 Thermal Cycler, Bio-Rad, T100 PCR Plate Sealer, Bio-Rad, PX1 1–10 µL single channel pipette...reagents and the NTC with virus. Pre-warm the 96-well plate sealer by gently touching the screen. Prepare the...the Serial Dilution Place a 48-well dilution plate in a chilled 96-well freezer block and place in the...

... We recommend that you plate 50 μL on one plate and the rest on a second plate. This gives the best chance...they will be able to grow once plated on the antibiotic containing agar plate. This step is not critical ...antibiotic resistances. Plate some or all of the transformation onto a 10 cm LB agar plate containing the appropriate...isn't too much liquid media on the agar plates. If the agar plate doesn't dry adequately before the cells... What went wrong? Check that you are plating on an LB Agar plate containing the correct antibiotic. The...tubes Sterile spreading device Reagents LB agar plate (with appropriate antibiotic) LB or SOC media Competent... on ice (approximately 20-30 mins). Remove agar plates (containing the appropriate antibiotic ) from storage...

...well plate sealer by gently touching the screen. Preparation of the Master Mix Place a ddPCR plate onto... Foil Heat Seal on the PCR plate with the red line facing up. If the plate sealer is not at temperature...place the PCR plate with the foil onto the metal support block. Place the block in the plate sealer and ...transfer the plate to the Droplet Reader. Open the QuantaSoft software to set up a new plate layout. Designate...Bio-Rad, DX200 Thermal Cycler, Bio-Rad, T100 PCR Plate Sealer, Bio-Rad, PX1 1–10 µL single channel pipette...PCR tubes, Axygen, PCR-02-FCP-C ddPCR 96-well PCR plates, Bio-Rad, 12001925 Pierceable foil heat seal, Bio-Rad...µL of each viral dilution to a well of a 6-well plate (1 dilution per well). Leave one well untransduced...

...standards and samples onto the qPCR plate Set up and load the 96-well plate: Load 5 μL of each standard in ... 58 °C 30 sec / read plate/ repeat 39x from step 3 / melt curve Example of plate set-up: 1 2 3 4 5 6 7...these institutions. This protocol is for a 96-well plate with 20 μL reaction volume. Last Update: February...Estimate of time required: ~3 h Workflow Timeline Plate set-up: 2 h qPCR run: 1.5 h Data analysis: 30 min...min Equipment qPCR instrument Heating plate Pipettors 1–10 µL single channel pipette 20–200 µL single ...Nuclease-free water Microcentrifuge tubes 96-well optical plate Pipette tips General Considerations Always run standards... the samples being tested. Always include a No Template Control (NTC), i.e master mix + water Whenever...

...If you are using the same template DNA for all your reactions, the Template DNA can be added to the master...particularly for GC-rich templates. What does each ingredient specifically do? Template DNA: Contains the portion...Taq Polymerase. From a single copy of DNA (the template), a researcher can create thousands of identical...polymerases from other species. In addition to the template DNA and the Taq polymerase, PCR requires free ...micrograms of target DNA from just a few nanograms of template DNA through several cycles of denaturation, annealing... initiation step heats the double stranded DNA template strand to the point where the strands start denaturing...range to anneal to each of the single stranded DNA template strands. The DNA polymerase is also stable enough...

...rocking platform. Permeabilize cells for 10 min at room temperature ( RT ) on a rocking platform in 500...rocking platform. Section 3: Labeling with antibody Block for 20 min at RT on a rocking platform in 500...Pipette controller Pipette tips and pipettes Rocking platform Tweezers Fluorescent microscope 0.5–10 µL single...Poly-D-lysine coated coverslips HeLa cells 24-well plate 4% Paraformaldehyde 5 mg/mL 4′,6-diamidino-2-phenylindole... in each well of a 24-well cell culture treated plate. Seed 5 x 10 3 HeLa cells per well. Allow the HeLa...cells Gently aspirate the media from the 24-well plate. Wash each well with 500 µL of PBS, remove the wash.... Wash 3x for 5 min in 500 µL PBS on a rocking platform. Dilute the fluorescently-labeled secondary antibody...

...cells, following manufacturer’s protocol. Plate on LB agar plates containing 100 μg/mL ampicillin or carbenicillin... transfected, plate 7×105 HEK-293T cells in 5 mL of media in a 6 cm tissue culture plate. Incubate cells...initiating your experiment. Day 1: a. Plate target cells in ten 6 cm plates and grow at 37°C, 5% CO 2 overnight...that you maintain one uninfected plate of cells in parallel. This plate will serve as a positive control...Particles This protocol is for transfection in a 6 cm plate. The protocol can be scaled to produce different...FBS with penicillin/streptomycin, cells should be plated at this step in DMEM + 10% FBS without antibiotics... you do not want to dislodge the cells from the plate. i. Incubate cells at 37°C, 5% CO 2 for 12-15 hours...

...Making LB Agar Plates Create plates to culture bacteria in the lab Pouring LB Agar Plates Protocol Streaking...Bacteria on Plates Isolate single bacterial colonies on an agar plate Streaking Bacteria on Plates Protocol...Antibiotic Plating Create plates to culture bacteria in the lab Over-Agar Antibiotic Plating Protocol AAV...

... a shaking platform. Wash the membrane 3x for 5 min in 1X TBST at RT on a shaking platform. Section 6:... a shaking platform. Wash the membrane 3x for 5 min in 1X TBST at RT on a shaking platform. Prepare the...supply iBlot 2 Gel Transfer Device Roller Spatula Platform shaker Cold room Gel imager -80 °C freezer Reagents... Tween-20 Nonfat milk powder 96-well microtiter plate Chemiluminescence substrate Plastic wrap Primary...into 200 µL of BCA reagent in a 96-well microtiter plate. Incubate for 30 min at 37 °C . Determine the absorbance...average absorbance of the duplicate samples on the plate. Subtract the average absorbance of the blank from...overnight in primary antibody at 4 °C on a rocking platform. Pro-Tip Primary antibody incubation can be reduced...

...week: Monday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM Complete. Wednesday: Plate 1×10 6 cells ...cells at 3.8×10 6 cells per plate in DMEM Complete in 10 cm tissue culture plates. Incubate the cells at 37...previously seeded 10 cm plate. Slowly pipette the transfection mix onto the 10 cm plate, being careful not ...controller Hazardous waste container pH meter Stir plate Magnetic Stir Bar Reagents DMEM high glucose, Corning... in a T75 flask in 15 mL DMEM Complete. Friday: Plate 8×10 5 cells in a T75 flask in 15 mL DMEM Complete...

...week: Monday: Plate 1x10 6 cells in a 75 cm 2 flask in a volume of 15 mL. Wednesday: Plate 1x10 6 cells...cells at 3.8x10 6 cells per plate in DMEM complete in 10 cm tissue culture plates. Incubate the cells at 37... 10 cm plate). 56.7 µL of 1 mg/mL PEI, MW 25,000 Da in 443.3 µL of OptiPro SFM per 10 cm plate Pro-Tip...cells in a T75 flask in a volume of 15 mL. Friday: Plate 8x10 5 cells in a T75 flask in a volume of 15 mL...

...+ insert plate should have significantly more colonies than the recipient plasmid alone plate. The recipient...you can expect on your recipient plasmid + insert plate that are not correct. If you have a high number ...number of colonies on your recipient plasmid alone plate, you can try ligating the recipient plasmid alone ...transformation worked. You should also verify that you are plating on the appropriate antibiotic and try varying the...the number of background colonies on your control plate (the more background, the more colonies you will...

...+ insert plate should have significantly more colonies than the recipient plasmid alone plate. The recipient...If you are amplifying from a plasmid or simple template, there is very little chance for mis-priming, ...you can expect on your recipient plasmid + insert plate that are not correct. If you have a high number ...number of colonies on your recipient plasmid alone plate, you can try ligating the recipient plasmid alone ...the number of background colonies on your control plate (the more background, the more colonies you will...

...Microcentrifuge Electrophoresis chamber Power supply Rocking platform Fume hood Metal spatula Razor blade Plastic tray... in-house standard Before Starting Warm the hot plate to 100 °C. Thaw IgG standard and prestained protein...water for 5 min with gentle agitation on a rocking platform. Pour off the water in the sink. Add 20 mL of ...incubate for 1 h with gentle agitation on a rocking platform. Pour off the SimplyBlue SafeStain in the sink...incubate for 1 h with gentle agitation on a rocking platform. Pour off the water in the sink. Add 100 mL of...incubate for 1 h with gentle agitation on a rocking platform. Pour off the water in the sink. Take a brightfield...

...correspond to sequences on the template molecule (must be complementary to template strand). However, primers...to design primers that are complementary to the template region of DNA. They are synthesized chemically...primers do not need to correspond to the template strand completely; it is essential, however, that the 3’...end of the primer corresponds completely to the template DNA strand so elongation can proceed. Usually ...

...your LB agar plates are not fresh, you should streak your bacteria onto a new LB agar plate before growing... Streaking and Isolating Bacteria on an LB Agar Plate Creating Bacterial Glycerol Stocks for Long-term...commonly used to culture bacteria in the lab. LB agar plates are frequently used to isolate individual (clonal...toothpick, select a single colony from your LB agar plate . Drop the tip or toothpick into the liquid LB +...

...new window) Link to Video Making LB Agar Plates Create plates to culture bacteria in the lab Watch the...Video! Over-Agar Antibiotic Plating Quickly add antibiotic to a pre-poured plate Watch the Video! Streaking...Bacteria Isolate single bacterial colonies on an agar plate Watch the Video! Inoculating a Liquid Bacterial ...

...with shaking platform set to 120 rpm 37 °C bead bath Vortex Stir bar Magnetic stir plate pH meter Reagents...water in a 1 L bottle and stir on a magnetic stir plate. Stir until all particles have dissolved. Note: ...flask.Incubate in a 37 °C, 5% CO 2 incubator on a shaking platform set to 120 rpm. Pro-Tip Do not use cells that ...

...days. Single cells were then plated in individual wells of a 96-well plate and left undisturbed for 13 ... well (each well gets one dilution, so a 6-well plate will hold 5 dilutions plus one 'no virus' control...