Esophageal Adenocarcinoma Methods and Protocols

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CAUTION! Conical flask may be hot when removing from
the microwave oven. Ensure that protective heat gloves are
worn when handling hot flask.


  1. Run a 5% agarose gel at 60 V for 150 min to determine
    primer specificity and identify the best annealing tempera-
    ture (see Note 9).

  2. Non-template control must be used in all assays to ensure that
    there is no cross contaminations (see Note 10).

  3. Run a second set of gradient ddPCR to select the final anneal-
    ing temperature.

  4. Prepare reaction mixture according to the manufacturer’s pro-
    tocol with consideration of pipetting error (see Note 11).

  5. For example, according to the ddPCR supermix protocol, we
    use a reaction mix. This contains 11 μL 2× ddPCR supermix
    for probes, 1 μL of 1 μM control gene primer set, 1 μL of
    1 μM target gene primer set, 1 μL of 2 U/μL restriction
    enzyme (which does not cut the amplicon of both the target
    gene and control gene) (see Note 12), and 30 ng of template.
    DNase-free water is added to the reaction mix to top up to a
    final volume of 22 μL.

  6. Transfer 20 μL reaction volume into the sample section of a
    microfluidic cartridge and subsequently with 70 μL of genera-
    tion oil to the oil section.

  7. Close the cartridge with a gasket and then place it into a drop-
    let generator.

  8. Carefully transfer all of the droplets into a 96-well plate (see
    Note 13) and seal it with a pierceable foil heat seal.


3.4 ddPCR Gradient
Optimization


Fig. 2 Internal hybridization parameters section for design of probes


Katherine T. W. Lee et al.
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