221- Non-template control must be used in all assays to ensure that
there is no cross contamination (see Note 7). - Top up with DNase-free water to a final volume of 20 μL.
- Amplify the sample to endpoint with a thermal cycler according
to the manufacturer’s protocol for the mastermix (see Note 8). - Store the amplicons in − 20 °C freezer until further use.
- Prepare a 2% agarose gel in TAE buffer (see Note 9) inside a
conical flask (see Note 10).
CAUTION! Conical flask may be hot when removing
from the microwave oven. Ensure that protective heat gloves
are worn when handling hot flask. - Add an intercalating dye such as ethidium bromide or Sybr
Safe for band visualization later.
CAUTION! Intercalating dyes are mutagens. Ensure pro-
tective wear such as gloves are worn and disposed of properly
after use. Be careful of vapors when adding dye to hot gel,
perform this step in a fume hood if adding dye to hot gel. - Load sample/DNA indicator ladder mixed with 2 μL of load-
ing dye to allow visualization of electrophoresis progression.
Perform electrophoresis at 70 V for 60 min (see Note 11). - Visualize sample using a gel imager (Fig. 1 ) or an ultraviolet
transilluminator.
CAUTION! When dealing with an ultraviolet transillumina-
tor, ensure that protective gear such as an acrylic shield or a face
shield is used to prevent unnecessary exposure to ultraviolet.
3.3 PCR Cleanup
Fig. 1 Example of gel image after a 2% agarose gel ran at 70 V for 60 min. Well
“1” represents a 100 bp ladder, wells “2 to 6” represent sample PCR products,
and “well 7” represents the non-template controlSingle Gene Mutation in Esophageal Adenocarcinoma