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20 EDVO-Kit # 953 Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants Notes to the Instructor: Gel Staining and Destaining AFTER ELECTROPHORESIS Instructor’s Guide After electrophoresis, the agarose gels require staining in order to visualize the separated DNA samples. This experiment features a proprietary stain called InstaStain®. InstaStain® EtBr (Appendix F) Optimal visualization of PCR products on gels of 1.0% or higher concentration is obtained by staining with InstaStain® Ethidium Bromide (InstaStain® EtBr) cards. Exercise caution when using Ethidium Bromide, which is a listed mutagen. Disposal of the InstaStain® EtBr cards, which contain only a few micrograms of ethidium bromide, is minimal compared to the large volume of liquid waste generated by traditional ethidium bromide staining procedures. Disposal of InstaStain® cards and gels should follow institutional guidelines for chemical waste. InstaStain® MetBlue: One-step Staining and Destaining (Appendix G) InstaStain® Methylene Blue (InstaStain® MetBlue) can be used as an alternative for staining gels in this experiment. However, InstaStain® MetBlue is less sensitive than InstaStain® EtBr and will yield variable results. Agarose gels can be stained and destained in one easy step, which can be completed in approximately 3 hours, or can be left in liquid overnight. For the best photographic results, leave the gel in liquid overnight. This will allow the stained gel to "equilibrate" in the destaining solution, resulting in dark blue DNA bands contrasting against a uniformly light blue background. Gels stained with InstaStain® Methylene Blue may be stored in the refrigerator for several weeks. Place the gel in a sealable plastic bag with destaining liquid. DO NOT FREEZE AGAROSE GELS! Used InstaStain® MetBlue cards and destained gels can be discarded in solid waste disposal. Destaining solutions can be disposed down the drain. Photodocumentation of DNA (Optional) There are many different photodocumentation systems available, including digital systems that are interfaced directly with computers. Specific instructions will vary depending upon the type of photodocumentation system you are using. Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2008, 2009 EDVOTEK, Inc., all rights reserved EVT 090205K The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Water Quality Testing III: Multiplex PCR Analysis for Water Contaminants EDVO-Kit # 953 21 Pre-Lab Preparations 1. Prepare the contaminated water samples: • Using a separate sterile loop for each culture, collect the cells by gently scraping the surface of each bacterial slant (B. subtilis, E. coli, S. marcescens). Draw the loop towards the top of the slant to collect the cells. • Transfer each culture into separate, labeled 50 ml conical tubes each containing 40 ml of distilled or deionized water. Use caution to avoid cross-contamination. • Mix the "contaminated water" for 15 minutes at room temperature. Alternatively, samples can be placed on a shaking platform. 2. Prepare the unknowns • Combine 5 ml of the E. coli and 5 ml of the S. marcescens and label this "Unk #1". • Prepare a mixture of any combination of the bacteria for unknown #2. Label this "Unk #2". Instructor’s Guide 3. Label and dispense 1.5 ml of each of the samples into 1.5 ml screw-cap tubes for each of the lab groups. 4. Resuspend each of the two tubes of Proteinase K with 50 µl of DNA Extraction buffer (H). Allow the Proteinase K to rehydrate for a minute and mix completely to dissolve. Each student group of five will need the following: 5 Tubes, each containing one of the contaminated water samples (in screw-cap tubes) 5 0.5 ml screw-cap tubes 1 Tube of Lysis Solution (0.6 ml), on ice 5 0.5 ml snap-top tubes 5 Tubes, each containing a PCR pellet 1 Tube of multiplex primer solution (130 µl) 1 Tube of Gel loading solution (50 µl) 1 Tube of 200 bp ladder (35 µl) 1 Tube of Potassium acetate (100 µl) 1 Tube of Tris buffer (150 µl) 5. Add all of the dissolved Proteinase K back to the remaining DNA Extraction buffer. Mix well and label “Lysis Solution”. Mix the Lysis solution between each aliquot and dispense 0.6 ml of Lysis Solution for each group. Keep on ice. 6. Dispense 130 µl of primer mixture (B) into labeled tubes, one per group. Keep on ice. 7. Aliquot 35 µl of 200 bp DNA ladder (C) into labeled tubes, one per group. 8. Dispense 150 µl of Tris buffer (E) into labeled tubes, one per group. 9. Dispense 100 µl of Potassium acetate (G) into labeled tubes, one per group. 10. Prepare 70% ethanol and isopropanol. Keep on ice. Divide students into four groups of five. Each student can work independently on one of the contaminated water samples. Designate one group to use the positive control DNA (D) for the control PCR reaction. Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2008, 2009 EDVOTEK, Inc., all rights reserved EVT 090205K The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Page 1 and 2: The Biotechnology Education Company
Page 3 and 4: Water Quality Testing III: Multiple
Page 19: Water Quality Testing III: Multiple
Page 27 and 28: Appendix A PCR Experimental Success
Page 29 and 30: Appendix C Preparation and Handling
Page 31 and 32: Appendix E 1.0% Agarose Gels - Quan
Page 33 and 34: Appendix G Staining and Visualizati
Page 35 and 36: Material Safety Data Sheets Full si

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