of the evidence DNA, the current son - Subject X’s DNA and the deceased son - Subject Y’s DNA. The PCR products you obtained with the first set of primers were labeled Evidence-1, X-1 and Y-1. The products obtained with the second set of primers were labeled Evidence-2, X-2 and Y-2. You now must run the agarose gel electrophoresis to examine the PCR products, compare the products from the subjects’ DNA and the products from the reference evidence, and determine whether your pride and joy is really true. Figure 6
BMES 221 - Engineering Principles of Living Systems I Procedure Procedure A: Cast Agarose Gel Prepare Agarose Gel a. dilute 10x TBE 1x TBE (Tri-borate-EDTA buffer solution). b. 1 Erlenmeyer flask contains agarose for 2 gels. c. Add 150 ml of 1x TBE. d. Put a paper towel plug into top of flask. e. Microwave till agarose is dissolved, around 2 minutes, CAUTION – HOT! f. Put flask into 37C water bath to cool. g. If you are adding Gel and Buffer Stain to your gel, measure 80 μ l of the Carolina BLU stain with the dropper bottle (2 drops) and swirl to mix it in the warm agarose. Make certain the agarose is cool enough to be comfortable on the inside of your writst before adding stain. h. Affix gel tray side-ways in gel box, rubber gasket against sides. i. Seal ends of gel-casting tray with tape, and insert well-forming comb. Place gel-casting tray out of the way on the lab bench, so that agarose poured in the next step can set undisturbed. j. When agarose is cool enough to hold in your hand, fill 2 gel trays with about 75 ml each. Carefully pour enough agarose solution into the casting tray to fill to a depth of about 6mm. The gel should cover about ½ the height of the comb teeth. Use the tip of a transfer pipet to move large bubbles or solid debris to the sides or the end of a tray while the gel is still liquid. k. Gel will become cloudy as it solidifies (about 10 minutes). Do not move or jar casting tray while the agarose is solidifying. l. Allow gel to cool for 30 minutes while you practice loading a gel. m. When agarose has solidified, remove tape from the ends of the casting tray and place the tray in the gel box so that the comb is at the negative (black) end. n. Fill the gel box with TBE buffer to a level that just covers the entire surface of the gel. o. Gently remove the comb, being careful not to rip the wells. One of you should hold the tray firmly while another gently pulls the comb straight up. p. Make certain that sample wells left by the comb are completely submerged. q. The gel is now ready to load with DNA.
BMES 221 - Engineering Principles of Living Systems I Procedure B: Load Gel Use a transfer pipet or a micropipette to load the contents of each tube of DNA (Evidence-1, Evidence-2, X-1, X-2, Y-1, Y-2) into separate wells, aligned as shown in figure 7. A fresh pipet or pipet tip is used for each tube.
- Spring '12
- DNA, Forensic DNA Fingerprinting