...solution later in this protocol. Isolating a Monoclonal Cell Population by Limiting Dilution Isolate cells...Protocols Cloning by Limiting Dilution Isolating a Monoclonal Cell Population by Limiting Dilution You may also...solution will be used to seed the 96-well plate. Sample Calculation First, use a hemocytometer to quantitate...cells/mL To seed one 96-well plate, make 10 mL of a 5 cell/mL solution. Calculate the total cells needed: ...into each well of a 96-well plate. By doing this, you are seeding the plate at an average density of 0.5... will help you focus the microscope later when scanning the plate for individual cells, which can be difficult...scan the plate for individual cells, which should be on the same focal plane. Scan the entire plate and make...

...number of plates you’d like to pour. For example: Because we’d like to make 20 plates, and our plates can hold... Protocols Pouring LB Agar Plates Pouring LB Agar Plates You may...Sterile H 2 O Sterile plates of your desired size - we usually use 60 mm x 15 mm plates which can hold 5-10...colonies, we recommend using larger plates. Many labs use 100 mm x 15 mm plates which can hold 10 - 15 mL of ...appropriate number of plates and stack them on your lab bench. Label the plates with the date and the ... the first plate to get a good idea of what that volume looks like in your particular plate. For the remainder...remainder of the plates, pour directly from the bottle. Pro-Tips Be sure to swirl your plates after pouring...

... and Isolating Bacteria on an LB Agar Plate You may also like... Making LB Agar Plates Bacterial Transformation... last section of the plate, to create streak #3. Incubate plate with newly plated bacteria overnight (... DNA from it, you will want to isolate an individual clonal population (single colony) of bacteria from...single colony from a freshly streaked agar plate to inoculate a bacterial culture for DNA purification ...explains how to isolate a single bacterial colony by streaking it onto an LB agar plate. Last Update: Feb... LB agar plate (with appropriate antibiotic) Bacterial stab Procedure Obtain an LB agar plate with appropriate... one edge of the plate. Then streak horizontal lines in another section of the plate, and then diagonal...

... Protocols Inoculating a Liquid Bacterial Culture Inoculating a Liquid... may also like... Streaking and Isolating Bacteria on an LB Agar Plate Creating Bacterial Glycerol Stocks...culture bacteria in the lab. LB agar plates are frequently used to isolate individual (clonal) colonies of ...necessary to isolate enough plasmid DNA for experimental use. The following protocol is for inoculating an overnight.... You can now isolate your plasmid DNA from the bacterial culture by following Isolating Your Plasmid ...your LB agar plates are not fresh, you should streak your bacteria onto a new LB agar plate before growing... for Inoculating a Bacterial Culture Protocols...

... on LB Agar Plate Over-Agar Antibiotic Plating You may also like... Making LB Agar Plates Bacterial Transformation...device that fits the plate. Incubate plates at 37 ℃ for 18 hours. Day 2 Observe plates for colony formation...concentrations of carbenicillin plated over-agar. Control Plate with No Carbenicillin Plate shows a lawn of E. coli...Carbenicillin plated over-agar Plate shows less individual colonies than the 1 mg/mL plate and effective...purpose, provided that they fit your petri plate. Incubate the plate at room temperature for at least 30 minutes...selection. 150 µL of 0.1 mg/mL Carbenicillin plated over-agar Plate shows a lawn of E. coli and no apparent...selection. 150 µL of 1 mg/mL Carbenicillin plated over-agar Plate shows several individual colonies and effective...

...overnight. Plate 2.5 mL of the transformed cells on each of the eight bioassay plates (two plates per tube...Antibiotic 245 mm bioassay plates (Molecular Devices, X602) Pro-Tip Pour these plates at least one day in advance... Agar + Antibiotic plates at 37 °C. Prewarm 8X LB Agar + Antibiotic Bioassay plates. Prechill Micropulser...100 dilution). Plate 100 µL of each dilution onto a prewarmed Petri dish. Incubate plates at 30 ℃ overnight... edge that can scrape plates more abrasively at a certain angle. Incubate plates upside down at 30 ℃ overnight... are not present on the most dilute plate, count the second plate (ie. the 1:10,000 dilution). After 12...LB to scrape bioassay plates and remove bacteria. Use one scraper for all plates. Use two 10 mL pipettes...

... forget to factor this into your calculations later (see calculation example below). Seed 300,000 cells...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... a CSV file which will be used to calculate the titer. Calculations In the experimental setup above, the...dilution into 6-well plate 2.5 25 5 50 10 100 20 200 40 400 80 800 160 1600 To calculate the titers, first...

...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...compliance with your institution’s biosafety regulations. Equipment Class II, Type A2 Biological Safety...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...

...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...

... 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...also make their own competent cells. This is a relatively simple procedure and is useful for performing...Watch the protocol video below to learn how to isolate single bacterial colonies. Equipment Shaking incubator...

...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... A.1 The RNAi Consortium A.2 Map of pLKO.1 A.3 Related plasmids B. Designing shRNA Oligos for pLKO.1 B...terminal repeat. RRE Rev response element. A.3 Related Products The following plasmids available from ... have at least 3 nucleotide mismatches to all unrelated genes. TIP: Addgene recommends that you select...

...window) Video Link Description Related Page Making LB Agar Plates Create plates to culture bacteria in the...the lab Pouring LB Agar Plates Protocol Streaking Bacteria on Plates Isolate single bacterial colonies ...Antibiotic Plating Create plates to culture bacteria in the lab Over-Agar Antibiotic Plating Protocol AAV...colonies on an agar plate Streaking Bacteria on Plates Protocol Diagnostic Restriction Digest Verify a plasmid...efficiency Bacterial Transformation Protocol Inoculating a Liquid Bacterial Culture How to prepare and...and grow bacteria in a liquid medium Inoculating a Liquid Bacterial Culture Protocol Getting Started with... an Agarose Gel Gel purification allows you to isolate and purify DNA fragments based on size. Following...

...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...amplifying for analysis and manipulation. It is essential that the template DNA and primer are complementary...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...

... from different cells in the population. Once the polyclonal populations are growing well and have been...Consider expanding populations transduced with a variety of dilutions and pick the population that has the ...days. Single cells were then plated in individual wells of a 96-well plate and left undisturbed for 13 ...a more homogenous (but still polyclonal) cell population. Depending on the transducibility of the cell...selection may be a vital step for obtaining a population of cells that have taken up the lentiviral transgene... well (each well gets one dilution, so a 6-well plate will hold 5 dilutions plus one 'no virus' control...be 10 µg/mL. Pro-Tip Transducing too many cells relative to the number of virus particles reduces the transduction...

...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...

...+ insert plate should have significantly more colonies than the recipient plasmid alone plate. The recipient...function in the same buffer, as this will save time later. In our example, we will use EcoRI and NotI to ligate...TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a final Reverse Primer sequence of 5’-...If you are amplifying from a plasmid or simple template, there is very little chance for mis-priming, ...from genomic DNA, a cDNA library, or by RT-PCR. Isolate your PCR product from the rest of the PCR reaction... used. Follow the manufacturer’s instructions. Isolate Your Insert and Vector by Gel Purification Run ... agarose gel and conduct a gel purification to isolate the DNA. When running a gel for purification purposes...

...+ insert plate should have significantly more colonies than the recipient plasmid alone plate. The recipient... as well as a specifically designed test digest later to verify that the insert was cloned in the correct... used. Follow the manufacturer’s instructions. Isolate your insert and vector by gel purification Run ... agarose gel and conduct a gel purification to isolate the DNA. When running a gel for purification purposes...base pairs for each varies, it is difficult to calculate this based on DNA concentration alone. One method...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 ...

...microtiter plate. Incubate for 30 min at 37 °C . Determine the absorbance at 590 nm . Calculate the average... 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...average absorbance of the duplicate samples on the plate. Subtract the average absorbance of the blank from...BSA standard concentration versus absorbance. Extrapolate the total protein concentration of the sample...

...Streaking Bacteria Isolate single bacterial colonies on an agar plate Watch the Video! Inoculating a Liquid Bacterial...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...your DNA sequence into a cell line Isolating a Monoclonal Cell Population by Limiting Dilution Generate monoclonal...Watch the Video! Purifying DNA from an Agarose gel Isolate specific DNA bands cut from a gel Watch the Video...

...Timeline Day 1: Run SDS-PAGE and stain gel Day 1 or later: Image analysis Video Watch this instructional video...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...