I am trying to run an agarose gel electrophoresis of qRT-PCR products amplified by SYBR Green Mastermix to confirm the primer specificity, could I directly load the products into the agarose gel stained with Redsafe or does the products need to be loaded with 6X loading dye?
The loading dye contains a heavy substance (usually glycerol, but sometimes Ficoll or similar) that will let your DNA sample sink though the buffer and settle into the bottom of the well. Without this, a lot of your DNA will diffuse into the running buffer and you won't see it on the gel. So in short, you do need to use it. If you have a good reason for wanting to avoid putting dye in your samples, you can find recipes online for dye-free DNA gel loading buffers. If you do this, I would highly recommend pouring a gel that has more wells than you need, and putting 1X loading dye without DNA into at least one well. This way, you can still track that your gel is running correctly, even though your samples contain no loading dye.
As suggested by Laura and explained it is appropriate to load using 6x Loading dye, and for better resolution of lower molecular weight DNA products use a 3.5% Agarose gel with syber safe. Also use a ladder which has 100 to 500bp.
Jack Yao You need to use 6X loading dye to settle the DNA in the bottom of the lanes.
- Badges
- Science topic
- Similar topics
- Geoscience
- Cartography
- Mapping