Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 27

Figure 4.21.  Load above the well, not allowing the tip to enter and possibly puncture the well. Photo by author. Figure 4.20.  Each DNA sample has to have enough loading dye in it so that it will sink into the well. If the loading dye is a 10X concentrate, that means 1 part loading dye is added for every 9 parts of sample. Photo by author. calculations and diagram the preparation of the gel. Once the gel is solidified, secured the gel tray gates, if they are present, in the down position. Place the gel tray in the gel box in the correct orientation with the sample wells adjacent to the negative electrode. Be careful to not let the agarose gel slide off the gel tray. Add the 1X electrophoresis buffer to the gel box, covering the gel about 5 mm. Gently remove the comb (under buffer) from the gel. Part II: Preparing and Loading the Samples 92 • Position the gel box where it will be “run.” Decide which samples should be loaded into which wells. • Each group may not be running exactly the same samples in their gels, depending on the available samples. In your notebook, draw a diagram of the gel to show the six samples to be loaded and volumes that you intend to load into each well of the gel. 3. Assemble all of the samples, in labeled 1.7-mL reaction tubes, to be loaded into the gel. If the samples do not already have DNA loading dye in them, add loading dye as described in the Materials List. 4. Practice loading 15 µL of practice loading dye (concentrated loading dye diluted to 1X with dH2O) into Lane 1 (see Figure 4.21). Flush the practice loading dye out with several pipetfuls of electrophoresis buffer. 5. Load 20 µL of a salmon testes DNA/loading dye sample into Well 1 of the gel. 6. Load 20 µL of an E.coli DNA/loading dye sample into Well 2 of the gel. 7. Load 7 µL of the Lambda/HindIII DNA/loading dye sizing standard markers into Well 3 of the gel. 8. Load additional samples already mixed with loading dye into the other 3 wells of the gel. Choose what samples to load depending on what is available. For example: - Load another salmon testes DNA sample - Load another E.coli DNA sample - Load a yeast DNA sample - Load a plasmid DNA sample - Load an uncut DNA sample - Load a plant DNA sample 9. Record on your loading diagram exactly what got loaded and into which well. Chapter 4   Laboratory Manual