Forensic DNA Fingerprinting (Week 2)

Transcription

1 Forensic DNA Fingerprinting (Week 2) Instructions The purpose of today s lab is to: separate fragments of DNA by agarose gel electrophoresis; stain the gels; and observe the banding patterns on the gels and use this information to identify the putative Pennypincher daughter. Material Needed Quantity (!) Electrophoresis Unit 1 " Agarose gel 1 " Digested DNA samples 6 " DNA markers (ladder) 1 " Pipette tips, µl 13 tips " Micropipette, µl 1 " Waste container 1 " FastBlast DNA Stain, 100x ~120 ml " Large containers for destaining 1 " Foam micro test tube holder 1 " Power supply 1 " Gel staining tray 1 " Electrophoresis buffer (1x TAE) ~275 ml " Activity 1. Separating Fragments by Electrophoresis Procedure 1. Obtain a prepared agarose gel. 2. Remove your digested samples from the freezer and thaw with your hands. Gently tap the tube on the surface of the lab bench to bring any drops in the bottom of the tube. 3. Using a new tip for each sample, add 5 µl of sample loading dye "LD" to each tube: DNA Samples Loading Dye EOP 5 µl C1 5 µl C2 5 µl C3 5 µl C4 5 µl C5 5 µl 4. Tightly cap each tube. Mix the components by gently flicking the tubes with your finger. To bring the contents to the bottom of the tube, gently tap the tubes on the bench top. 97

2 5. Place the casting tray with the solidified gel in it, into the platform in the gel box. The wells should be at the ( ) electrode end of the box, where the black lead is connected. 6. Ask your instructor to pour ~275 ml of electrophoresis buffer into the electrophoresis chamber. He will pour buffer in the gel box until it just covers the gel and enters the wells. 7. Obtain the tube of DNA marker. The loading dye has already been added by your instructor. 8. Using a separate pipette tip for each sample, load your digested DNA samples into the gel. Gels are read from left to right. The first sample is loaded in the well at the left hand corner of the gel. Your instructor will do the first on for you so you can see it is done properly. Lane 1 DNA Size Marker (MK) clear tube 10 µl " loaded Lane 2 Pennypincher (EOP) green tube 20 µl " loaded Lane 3 Candidate 1 (C1) blue tube 20 µl " loaded Lane 4 Candidate 2 (C2) orange tube 20 µl " loaded Lane 5 Candidate 3 (C3) purple tube 20 µl " loaded Lane 6 Candidate 4 (C4) pink tube 20 µl " loaded Lane 7 Candidate 5 (C5) yellow tube 20 µl " loaded 9. Secure the lid on the gel box. The lid will attach to the base in only one orientation: red to red and black to black. Connect electrical leads to the power supply. 10. Turn on the power supply. Set it for 100 V and electrophorese the samples for min. While you are waiting for the gel to run, you may begin the review questions on the following page. 11. When the electrophoresis is complete, turn off the power supply and remove the lid from the gel box. Carefully remove the gel tray and the gel from the electrophoresis chamber. Place the gel and casting tray on a paper towel. Be careful, the gel is very slippery! Proceed to the next section for detailed instructions on staining your gel. Answer Questions 1-4 on the Lab Report. Activity 2. Staining DNA with Fast Blast DNA Stain Since DNA is naturally colorless, it is not immediately visible in the gel. Unaided visual examination of the gel after electrophoresis indicates only the positions of the loading dyes and not the positions of the DNA fragments. DNA fragments are visualized by staining the gel with a blue stain called Fast Blast DNA stain. The blue stain molecules are positively charged and have a high affinity for the DNA. These blue stain molecules strongly bind to the DNA fragments and allow DNA to become visible. These visible bands of DNA may then be traced, photographed, sketched, or retained as a permanently dried gel for analysis. 98

3 WARNING Although Fast Blast DNA stain is nontoxic and noncarcinogenic, latex or vinyl gloves should be worn while handling the stain or stained gels to keep hands from becoming stained blue. Take special care not to get the stain on your clothes! Procedure 1. Stain gels To remove each gel from the gel tray, carefully slide it into the staining tray. Pour approximately 120 ml of 100x stain into the staining tray. If necessary, add more 100x stain to completely submerge the gels. Stain the gels for 3-4 min, but not for more than 4 min. Using a funnel, pour the 100x stain back into the storage flask and save it for future use. The stain can be reused at least 7 times. 2. Rinse gels Transfer the gels into a large container containing ml of clean, warm tap water. Gently shake the gel in the water for ~10 secs to rinse.2 3 minutes. 3. Wash gels Transfer the gel into a large container with ml of clean, warm tap water. Gently swish the gels in the water once every minute. Students usually find that this works best by simply standing at one of the sinks in the lab and gently flooding the gel with warm tap water continuously. 4. Wash gels Perform a second wash as in step Record results Pour off the water and examine the stained gels for expected DNA bands. The bands may appear fuzzy immediately after the second wash, but will begin to develop into sharper bands within 5 15 minutes after the second wash. This is due to Fast Blast stain molecules migrating into the gel and binding more tightly to the DNA. Answer Questions 5-8 on the Lab Report. 99

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5 Forensic DNA Fingerprinting (Week 2) Lab Report Name Activity 1. Separating Fragments by Electrophoresis 1. The electrophoresis apparatus creates an electrical field with positive and negative poles at the ends of the gel. DNA molecules are negatively charged. To which electrode pole of the electrophoresis field would you expect DNA to migrate? (+ or -)? Explain. 2. What color represents the negative pole? 3. After DNA samples are loaded into the sample wells, they are forced to move through the gel matrix. What size fragments (large vs. small) would you expect to move toward the opposite end of the gel most quickly? Explain. 4. Which fragments (large vs. small) are expected to travel the shortest distance from the well? Explain. 101

6 Activity 2. Staining DNA with Fast Blast DNA Stain 5. On the figure below, sketch out the bands from your gel. 6. Label the top of the gel with each of the names of the sample that was loaded in each lane. 7. The bands of the DNA ladder are the following sizes: 23130, 9416, 6557, 4361, 2322, and Label the ladder sizes along the left margin of the gel. 8. Based on your data, who (if anyone) is the lost Pennypincher daughter? 9. What are the sizes of the fragments Pennypincher and his daughter have in common? 102