2254 Notes
- PCR hood should be exposed to ultraviolet light for 25 min
prior to using it. - Master reaction mixture is all the required ingredients to run
a PCR reaction excluding the template. For example, for 2×
PCR mastermix protocol, prepare 10 μL 2× mastermix, 1 μL
of 10 μM gene forward primer, and 1 μL of 10 μM gene
reverse primer. - Prepare an extra reaction for every eight reactions in consider-
ation of pipetting error and evaporation. - For every sample, three technical replicates are
recommended. - Three biological samples in duplicates (six reactions in total)
with one non-template control and one extra reactions. - It is recommended to use between 50 and 500 ng of template
when working with genomic DNA. - Non-template control is prepared by replacing the template
with DNase-free water. - Set the lid temperature to 105 °C or closest temperature avail-
able on the PCR machine used. - For every 100 mL of 1× TAE buffer, dissolve 2 g of agarose
powder. Ensure that agarose powder is completely dissolved
by heating in bursts of a few seconds and agitating flask at each
cycle. 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. - Using conical flask helps prevent overflowing of agarose liquid
when heating it in the microwave oven. - Routinely check the electrophoresis run to ensure that the
band does not run over the gel. - Protocol may vary from manufacturer to manufacturer.
- Usage of DNase-free water rather than the provided elution
buffer can help improve Sanger sequencing result. In addition,
total volume of DNase-free water used during elution can be
reduced if a higher concentration of elute is desired. - Optimal primer annealing temperature for Sanger sequencing
protocol should be around 60–65 °C. - Prepare an extra 10% of additional volume to compensate for
pipetting error and evaporation. - If bubbles are present at the top of the well, or in small quanti-
ties, it will not adversely affect the reaction.
Single Gene Mutation in Esophageal Adenocarcinoma