2074 Notes
- Ensure that there is no folding of tissue when placing the sec-
tion onto the slide. Also, ensure that the section is a 5 μM
thick section. This is to ensure proper staining and visualiza-
tion of the section. - Ensure that all buffers are topped up with the appropriate sol-
vents required. - Use DNase-free water to elute DNA instead of the provided
elution buffer. Add the recommended amount of DNase-free
water according to the manufacturer’s protocol and incubate
the column on the bench for 5 min before eluting the DNA. - Use DNase-free water as blank.
- Samples with low A 260 /A 280 ratio may have protein contami-
nation while higher A 260 /A 280 ratio may have RNA contami-
nation. Low A 260 /A 230 ratio may indicate residual (such as
phenol) carryover while high A 260 /A 230 ratio may indicate a
dirty blank used. Read the manufacturer’s troubleshooting
guide to help solve these issues. - Storing at − 20 °C prevents evaporation of sample over time
and ensures sample integrity. - Using conical flask helps prevent overflowing of agarose liquid
when heating it in the microwave oven. - 5% agarose gel is very concentrated and viscous and can solid-
ify in a very short time. After the initial 2 min microwave oven
heating, repeat the heating at 1 min interval until all agarose
powder is fully dissolved. Floating pieces of gel observed in
the flask is an indication that the agarose gel is not completely
melted. Once the gel is fully dissolved, quickly but carefully
transfer the solution onto the agarose gel cast. Ensure that all
bubbles are removed from the gel and leave to solidify. - Routinely check the electrophoresis run to ensure that the
band does not run over the gel. - Non-template control is prepared by replacing the template
with DNase-free water. - Prepare 22 μL of reaction mix instead of 20 μL in consider-
ation of pipetting error and evaporation. - A restriction enzyme is added into the ddPCR reaction mix-
ture to help reduce noise such as nonspecific amplifications
and thus creating a much cleaner data. - Slant the cartridge at approximately 30°. Place the micropi-
pette tip vertically into the well and slowly draw up the
droplets.
DNA Copy-Number in Esophageal Adenocarcinoma