Genetics Workshop: PCR-based Testing for Bacterial Contaminants in Water Samples. July 23,2007 Little Big Horn College Crow Agency, MT.

Transcription

1 Genetics Workshop: PCR-based Testing for Bacterial Contaminants in Water Samples July 23,2007 Little Big Horn College Crow Agency, MT Lab Manual 1

2 Water Quality Testing PCR-based Testing for Water Bacterial Contaminants Experiment Objective: The objective is to use PCR to determine the quality of water and to detect and monitor for the presence of bacterial contaminants in water samples. Overview of Experiment: 1. Isolate bacteria from the water sample by centrifugation 2. Isolate bacterial DNA 3. Prepare samples for PCR reactions 4. Run PCR reactions 5. Prepare agarose gel for electrophoresis 6. Load agarose gel 7. Run gel 8. Stain gel 9. Photograph gel for documentation 10. Analyze results, trouble shoot and plan for next experiment Materials Needed: (Provided by EDVOTEK, The Biotechnology Education Company Equipment: Thermal cycler (eg. Edvotek Cat #532) Horizontal Gel electrophoresis apparatus D.C. power supply Balance Microcentrifuge Photo documentation set up (Polaroid or Digital) Safety googles Automatic pipetors (5-50ul) Water bath Hot plate or heating block Vortex Disposable pipetors 250 ml flasks and other glassware Disposable gloves Hot gloves Microwave oven Ice buckets and ice Distilled water 2

3 Reagents and Supplies (comes with Edvotek kit) Agarose Electrophoresis Buffer CarolinaBlu loading Gel solution Microcentrifuge Tubes with screw caps (0.5 ml and 2 ml) PCR tubes E. coli specific primers E. coli DNA for positive control Chelating agent Tubes with PCR reaction pellets including, dntp mixture, Taq polymerase, Taq polymerase buffer, MgCl2 Proteinase K E. coli slant for water contamination Tris Buffer Sterile Water Plan: Water samples will include water spiked by the instructors with E. coli, control samples, and water collected from sources around LBHC and Crow Agency. The class will divide into pairs and will be given two samples per group to prepare for DNA extraction, PCR and gel electrophoresis. Background information (from EDVOTEK, The Biotechnology Education Company ( 3

4 Methods and Procedures A. Isolation of Bacteria from Water Samples 1. Take tube of the water sample. 2. Label tube with initials of one member of your group. 3. Spin the tube at max speed for 15 minutes to pellet the bacteria. 4. Remove supernatant (liquid) with a pipet and place it in a separate tube. Write your observations of the pellet here and compare notes with other groups 5. Add back 175 µl of supernatant to the pellet and resuspend by vortexing. 6. Discard the unused supernatant in the biological disposal container. B. Isolation of Bacterial DNA for PCR 1. Mix the tube containing the lysis solution (buffer, chelating agent + proteinase K) by inverting several times. 2. Continue to mix the lysis solution with a pipet by pipetting up and down several times. 3. Then quickly add 50 µl of the lysis solution to the tube containing the resuspended pellet from step A.5 above. 4. Vortex the tube or mix by inverting several times. 5. Place samples in 56 C heating block for 30 minutes. (This step will digest the bacterial cell wall and release the DNA). 6. Place samples in 95 C heating block for 10 minutes. (This step will destroy the Proteinase K) 7. Allow samples to cool to room temperature for 2 minutes. 4

5 8. Spin the tubes at maximum speed in microcentrifuge for 5 minutes. 9. Remove supernatant and place in a clean tube. Place tube on ice. Make sure that chelex (beads at bottom of tube) is not transferred to the PCR tube. This will inhibit PCR process. If there are beads present, do a second spin in the microcentrifuge by repeating step Dilute your bacterial DNA 1:100 before you begin PCR by adding 10µl of the DNA sample from step 9 to 990µl of distilled water in a fresh tube. 11. Vortex briefly and store on ice. NOTE: This isolated bacterial DNA can be frozen or used immediately for PCR. Today, we will go onto Part C right away. C. Prepare samples for PCR 1. Label a PCR tube with a number and the initials of one member of your group. The PCR tube will have a small white bead inside that contains the taq polymerase, dntp s, salts and buffer (bead will dissolve when primers are added) 2. Tap the reaction tube to assure that the reaction pellet is at the bottom of the tube. 3. Add the following to the PCR reaction tube 20 µl of the primer mix. 5 µl of DNA isolated from Part B above (vortex briefly first) 4. Instructor (or students) will prepare control PCR reactions as follows: Negative Control - using distilled water in place of DNA (20 µl of the primer mix + 5 µl of distilled water to a beaded PCR tube). Positive Control using E. coli DNA (20 µl of the primer mix + 5 µl of E. Coli DNA) 5. Mix the tubes gently by tapping, then place in the microcentrifuge and spin a few seconds to bring contents to the bottom of the tube. Make sure the reaction pellet is completely dissolved. 6. Place the tubes in the thermal cycler. 7. Once all students have inserted tubes, close the lid and press start. It will take a few minutes for the heated lid to warm up. 5

6 8. Observe thermal cycler screen through one full cycle to confirm that it is running through the correct cycling program as follows (this was programmed by your instructor). PCR reactions Step1 95 C for 5 min. (initial denaturating) Step2 30 cycles of: 94 C for 45 sec. (denaturating) 50 C for 45 sec. (primer annealing) 72 C for 45 sec. (extension of PCR product) Step3 72 C for 7 min (final extension) NOTE: Samples are ready to load for gel electrophoresis or can be stored in the refrigerator for later use. 6

7 C. Preparing Agarose (Instructors may have completed this Part) 1. Use a 250 ml flask to prepare the gel solution. Add the following components to the flask to make a 1% Gel Amt of Agarose 50X Buffer DiH20 Total Volume (gm) (ml) (ml) (ml) for 7X7 gel for 7X14 gel Swirl the mixture to disperse clumps of the agarose. 3. Mark the level of the solution on the outside of the flask. 4. Heat the mixture in a microwave oven until dissolved. The final solution should appear clear without particles. Cover the flask with plastic wrap and heat on high for 1 min. Remove and swirl the mixture, then heat again in bursts of 25 secs until dissolved. 5. Cool the agarose to 55 C with careful swirling. If evaporation has occurred, add dih20 to bring to the original volume marked on the flask. 6. Store in 55 C heated water bath until ready for Part D to prevent congealing. D. Gel Electrophoresis 1. Prepare casting tray for gel as follows: -if using Cabisco biotechnology gel box (clear) seal the ends of the tray using tape. -if using Owl gel box (yellow) place the tray inside the gel box so that the red gaskets seal against the yellow sides, and push down until it touches base at all points. 2. Place the well-forming combs in the slots, pushing both sides as far as they can go. 3. Take one bottle of melted agarose from the heated water bath. (OPTION: Add 2 drops of CarolinaBlu Gel and Buffer Stain to the gel and quickly swirl to mix). 4. Pour the gel slowly into the casting tray until it reaches 1/3 of the way up the comb teeth (this may not require all of the gel solution). Allow the gel to solidify for 20 minutes. During this time, participants should practice filling wells on the provided practice gel using the blue loading dye. 7

8 5. Carefully remove the combs from the gel, holding on both sides and pulling up slowly and evenly. 6. Remove the tape (Cabisco) or pull the gel out of the box (Owl) and place in the gel box with the wells at the end with the red dot. 7. (OPTION: Pour 1X TBE containing CarolinaBlu into the gel box just until the gel is completely covered.) 8. Load the gel and record what is in each lane. Add lanes to the diagram as needed. Lane 1 Ladder 9. Place the lid on the gel box, aligning the electrodes over the pegs and pushing in firmly. 10. Plug the other end of the electrodes into the power supply in the correct orientation. 11. Turn on the power supply and set to medium if using small power supply or set voltage to 100V on the larger power supply. Allow to run for approximately 45 minutes. You can check the gel periodically to see bands migrating through gel. 12. Turn off the power supply and remove the electrodes from it. 8

9 13. Remove lid from gel box and remove the gel, keeping it on the tray and place on a paper towel. 14. Mark your gel by cutting off a corner in a way that you will recognize later using a spatula. 15. Slide gels from casting tray into the clear pyrex dish and place on a flat surface covered with plastic wrap. (OPTION: cover with the CarolinaBlu post-stain solution). 16. Wearing gloves, place the blue side of the InstaStain card on the gel (OPTION: After 30 minutes pour off the CarolinaBlu into the original container. Save this solution as it can be used several times). 17. Firmly run your fingers several times over the entire surface of the card to establish good contact between the card and the gel. Place a weight on the card to insure continuous contact. (OPTION: Cover the gels with distilled water and allow to sit for 10 minutes. Pour water into waste container and repeat wash two more times or until bands can be seen clearly). 18. Allow the InstaStain to sit on the gel for 5 10 mins. 19. Remove the card. If the color is very faint, wet the gel surface with dih20 and replace the card for 5 mins. 20. Transfer the gel to a container and destain with dih20 until bands are clear. 21. Instructors will demonstrate how to take pictures of the gels. 22. Analyze bands on the gel and draw them on the gel image on the previous page. Write a summary of your conclusions and observations here: 9