Step By Step description how to prepare Agarose gel and to use dsDNA binding dye like dsGreen or SYBR Green for DNA detection by gel soaking.
We recommend the Gel soaking method to be used with dsGreen by Lumiprobe® or with SYBR® Green by Molecular Probes or other dsDNA binding benzothiazole dyes. Some difference between different possible staining approaches is described in in this article.
To prepare approx. 100g of 1,5% agarose gel measure 1,5g of stock agarose.
Mix agarose powder with 100mL 1xTAE buffer.
Microwave for 1-3 minutes until the agarose is completely dissolved. And let agarose solution cool down till you can comfortably keep your hand on the flask.
Pour the agarose into a gel tray with the well comb in place. Wait till the gel will completely solidified.
Remove well comb from the gel.
Place the agarose gel into the gel box. Fill gel box with 1xTAE until the gel is covered.
Carefully load a molecular weight ladder into the first lane of the gel. Carefully load your samples into the additional wells of the gel.
Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel.
Remember about gel orientation (Black electrode is negative, red is positive. The DNA is negatively charged and will run towards the positive electrode.)
Add 20 μL of Lumiprobe® dsGreen dye to 20 mL of 1xTAE buffer.
Place the gel into a container filled with prepared solution. Incubate 10-15 min at room temperature.
Visualize your DNA fragments in UV-light transilluminator.
Fragments of DNA are separated in the gel depending on their length (smaller molecules travel faster than larger molecules in gel).
SYBR® is a registered trademark of Molecular Probes.