Introduction: Gel electrophoresis is an important molecular biology tool: it enables us to study DNA. It can be used to determine the sequence of nitrogen bases, the size of an insertion or deletion, or the presence of a point mutation; it can also be used to distinguish between variable sized alleles at a single locus and to assess the quality and quantity of DNA present in a sample. Gel electrophoresis is a method of separating chemical compounds and molecules by their size and charge. The substances being separated are placed in wells in an agarose gel and subjected to an electrical field.
Negatively charged molecules move towards the positive anode, and positively charged molecules move towards the negative anode. Longer or larger molecules have difficulty traveling through the gel; they become entangled in the gel matrix. Shorter or smaller molecules migrate through the agarose matrix faster and thus travel farther in a given time period. Similar sized fragments travel at relatively the same speed and form a tight “band” when stained. Pre-Lab Questions: DNA fingerprinting is the identification of DNA through the process of gel electrophoresis.
PCR is performed in order to amplify a specific section of DNA and is especially helpful when looking for one trait in particular. All possible results are: a) only one child has the gene for colon cancer, b) the twins have the gene for colon cancer c) all of the children have the gene for colon cancer, d) none of the children have the gene for colon cancer. Hypothesis: (See third pre-lab question) Materials: Pipettes DNA Samples Gel Gel Tray Masking TapeCarolinaBLU™ dye 1xL BufferGel Chamber Power SupplyDistilled Water Procedure: Tape the open ends of the gel tray using masking tape.
Run your thumb firmly along the taped edge of the tray to ensure a reliable seal. Insert the well-forming comb in the set of grooves at the end of the tray. Pour the gel into a depth of no more than 0. 5 cm deep. Pouring the gel thicker reduces the speed at which the gel can be run. While the gel is still liquid, move large bubbles and debris to the side using a pipette tip. Allow for the gel to solidify. Do not disturb the gel during the 10-15 minutes it takes for this to happen. The gel will look cloudy once it hardens. Place the gel in the chamber so that the wells are closest to the black negative electrode and cover it with 1x LB Buffer.
The layer of buffer over the gel should be no more than 1-2 mm deep. This shallow depth is another factor critical in allowing the gels to be run at a high speed. Load 20 ? L of each of the six samples from left to right, from sample A-E. To load each sample, place a clean, unused pipette tip on the end of the syringe. There should be a small piece of silicon tubing fitted over the end of the syringe on which you place the tip. This piece of flexible tubing creates a necessary seal between the pipette tip and the end of the syringe. Be aware that a relatively small movement of the syringe plunger is required to draw 20 ?
L into the pipette tip. The figure indicates the graduation on the yellow pipette tip that corresponds to 20 ? L. Once all of your samples are loaded, place the lid on the gel chamber. Connect all of the leads to the power supply (red to red and black to black) and set to the voltage indicated by your instructor. Once the orange loading dye has reached the end of the gel, turn off the power supply, disconnect the leads, and gently move your gel from the electrophoresis chamber to your straining tray. BE aware that the buffer will be very warm. Pour the CarolinaBLU™ final stain into the staining tray to just cover the gel.
Keep the gel in the stain for 20 minutes. Do not allow the gel to sit in the stain for longer than 45 minutes, or it will be hard to destain. Pour the CarolinaBLU™ stain back into the bottle, rinse the gel with distilled or deionized water, and fill the tray with just enough distilled water to submerge the gel. Further destain the gel with a couple more changes of distilled or deionized water at 15 to 30 minute intervals (again, just deep enough to submerge the gel). Alternatively, the gel can be left overnight to destain. The DNA bands will reach maximum visibility after from 5 hours to overnight.
Do not destain with tap water or with too much water, or the bands will fade. (In tap water, chlorine ions cause the fading. ) Results: Child B has the gene for colon cancer. The other two do not. Post-Lab Questions: One of the children carries the same mutation as the father (B). The child is likely to develop colon cancer. The twins are childs A and C due to their DNA being identical. Conclusion: Gel electrophoresis is used to separate genes by their size and is used in DNA fingerprinting and early detection of diseases such as cancer. It is a long, tedious process, but fascinating in the way it works.