Medbed: Teachers Prep for PV92 PCR Cheek Cell Protocol 7-14-10

Medbed: Teachers Prep for PV92 PCR Cheek Cell Protocol 7-14-10

MEDBEd: Teachers Prep for PV92 PCR Cheek Cell Protocol 7-14-10

Note to Teachers: Excluding the preparation of the agarose gels and any prelab/postlab activities/discussion, this activity is normally a three period or three day lab activity. It is a multistage lab with reagents that are time sensitive and require a minimum two hour amplification in the thermal cycler. In a research lab, you would add the primers and master mix and then immediately place the samples into the thermal cycler. However, it might be possible to have a class set of DNA sitting on ice, waiting for the next class period to complete the activity, before you place the samples into the thermal cycler, but it isn’t the suggested protocol. We suggest that you pick one class period for your first try.Our MEDBEd and Bio-Rad instructions are for one class of up to 32 students using one PV92 kit. Since MEDBEd provided only 3 electrophoresis chambers, we suggest dividing your class into 6 teams with 2 teams sharing each chamber.

_____ 1. Email MEDBEd at or call the MEDBEd office at 918-595-4611 to schedule delivery of the thermal cycler to your school.

_____ 2. Gather together the following documents and make copies as needed:

A.Pre-tests and Post-tests (MEDBEd provided 60 of each of these with large clasp envelopes addressed to TCC. Please print more as needed. The students will take the pre-test BEFORE they see the manuals and BEFORE any pre-lab instructions. Remind the students that they are not expected to do well on the pre-tests and that the same questions will reappear later as a post-test after the lab is completed.)

B.Bio-Rad “Biotechnology Explorer, Chromosome 16, PV92 PCR Informatics Kit, Instructor’s Manual (This is the manual that came with your PV92 kits at the MEDBEd Workshop #3. In addition to basic lab prep instructions, the manual includes good background information.)

C.MEDBEd PV92 PCR … Items Needed for Lab (This list includes MEDBEd items as well as some items that need to be provided by your school.)

D.Power Supply/Electrophoresis Chamber Instructions Summary

E.Bio-Rad MyCycler Thermal Cycler Instructions Summary

F.Bio-Rad Student Instruction Manuals (The Bio-Rad Student Manual begins on page 39 of the Bio-Rad Instructor’s Manual. The Student Manual includes some good background information. From the Student Manual pages, choose the pages you wish to duplicate for your students. You used the cheek cell protocol in the MEDBEd Workshop.)

G.PV92 PCR Student Instructions and Checklist This document provides the basic lab instructions for students ANDALSO details how to make and store gels, how to aliquot the loading dye and DNA, how to use the microcentrifuge, how to use the micropipets, how to load the gels, how to place the gel tray into the electrophoresis chamber, how to stain and destain the gels, and much more. In addition, it gives reasons for usingloading dye, explains why the DNA migrates across the gel, etc.

H.Student Workstation Inventory, Day #1, Day #2, and Day #3 (optional documents)

I.TCC Model Release (Make copies of this document if you wish to share photos of your students.)

_____ 3. Visit the Bio-Rad website to see the power point teaching resources for PV92 PCR. Upon your request, MEDBEd can email the webite address to you so that you can click on the link. You may wish to use some of the power point slides with your students.

_____ 4. Gather together the necessary equipment listed on the document, “MEDBEd PV92 PCR …Items Needed for Lab.”

_____5. Prepare the TAE buffer. Label an empty gallon size distilled water jug, “1x TAE” with the date. To make 3 liters of 1x TAE buffer, add 60 mL of 50x TAE to 2.94 L (2940 mL) of DI water or distilled water. Any TAE buffer remaining in the chamber after electrophoresis, may be returned to the TAE jug for reuse.

_____6. Prepare the agarose gels. To save classtime, some teachers prepare the gels for the students. Other teachers want the students to prepare their own gels. To make gels, see the directions on page 2-3 of the “Student Instructions and Checklist.” Use the combs that make 15 wells. Many teachers prefer to share each gel between two teams and since MEDBEd provided only 3 electrophoresis chambers, you may wish to divide each class into 6 teams sharing 3 gels/chambers.

_____ 7. Prepare 71% alcohol to be used for cleaning work areas. Mix 390 mL of 91% isopropol alcohol with 110 mL of DI water or distilled water. (Both 70% and 91% concentrations are readily available at your local pharmacy.)

_____ 8. For 2 minute staining, called quick staining, prepare the 100x Fast Blast stain. Mix 100mL of 500x Fast Blast stain with 400 mL of DI water or distilled water. Close and label the container. Store at room temperature until ready to use. This stain can be reused multiple times.

_____ 9. Check the supply of micropipette tips for each team.

_____ 10. Final set up for Day #1 or Lesson #1 Lab:

  1. Aliquot the InstaGene matrix as follows: Get the stock container from your refrigerator. While aliquotting, thoroughly mix by vortexing the InstaGene matrix to resuspend the matrix. Beads settle quickly, so remix FREQUENTLY while aliquotting. Aliquot 200µL InstaGene into one screw cap tube per student. Place all of the tubes into one plastic baggie and label the baggie, “InstaGene Matrix…1 tube per student.”Refrigerate until ready to use.
  2. Set your water bath to 56oC. It may take several hours to reach the correct temperature, so allow plenty time.
  3. Use your hot plate, thermometer, and a beaker/pan, to create a second water bath at 100oC. You will need enough floating space in your water bath for 6 foam test tube holders, one holder per team.
  4. Prepare the saline solution. Add 4.5 grams of table salt without iodine, to a 500 mL (16.9 oz) bottle of drinking water. Cap and shake the bottle until the salt is dissolved. Pour approximately 10 mL of saline solution into one clean cup per student.
  5. Set up 6 student workstations as listed on the Student Workstation Inventory for Day #1.
  6. For a review on how students should use the micro centrifuge machine and how to use the adjustable micropipets, please refer to Lesson #1, on pages 4-5 of the MEDBEd/SEEDBEd “PV92 PCR Student Instructions and Checklist” document.
  7. Refer to the Bio-Rad Student Manual and the MEDBEd/SEEDBEd “PV92 PCR Student Instructions and Checklist” document for the Lesson #1 lab procedures for students.

_____ 11. Final set up for Day #2 or Lesson #2 Lab:

  1. From your freezer, get one vial of each of the following: master mix, primer mix, +/+ control, -/- control, and +/- control. Allow the reagents to thaw in the refrigerator or in an ice bucket. After thawing, spin briefly in the micro centrifuge before opening vials.
  2. Since each gel will be shared by 2 teams, only one set of controls is needed per gel. With a fine point permanent marker, label the top and side of9 PCR tubes as follows: Label 3 tubes “+/+” Label 3 tubes “-/-“ Label 3 tubes “+/-“ Aliquot the controls as follows: Pipet 20 µL of the +/+ control into each of the +/+ tubes, Pipet 20 µL of the -/- control into each of the -/- tubes, and Pipet 20 µL of the +/- control into each of the +/- tubes. Store these control tubes in the refrigeratoror on ice until ready to use. Refreeze the remainder of any unusedcontrols in their original vials for future use.
  3. Review the “PV92 PCR Instruction Summary for Bio-Rad MyCycler THERMAL CYCLER” document provided by MEDBEd and get the thermal cycler set up, plugged in, and ready.
  4. Set up 6 student workstations as listed on the Student Workstation Inventory for Day #2.
  5. Label six empty snap cap micro test tubes, “mm,” and place tubes in a baggie or foam test tube holder. Store empty tubes in refrigerator or on ice.
  6. IMMEDIATELY BEFORE LAB OR NO MORE THAN 30 MINUTES BEFORE THE PCR AMPLIFICATION PROCESS, prepare the complete master mix by adding primers.These instructions are for one class of 17 to 32 students.(For a class of up to 16 students, cut the following amounts in half and refreeze the unmixed primer and master mix for future use.) Aliquot 1100 µL of master mix from the vial into an empty snap cap micro test tube. Add 22 µL of the primer mix to the 1100 µL of master mix. Vortex at least 10 seconds to mix. The solution should be yellow. Aliquot 180 µL of the complete master mix into each of the 6 snap cap tubes labeled, “mm.” Place one complete master mix tube into each team’s ice bucket. Please note: This 180 µL should be adequate for at least 5 team members at 20 µL each and for 3 controls at 20 µL each.
  7. During the lab, while the students are each transferring 20 µL of the supernatant to their own PCR tubes and adding 20 µL master mix,DESIGNATE 3 OF THE STUDENT TEAMS (ONE TEAM PERSHARED GEL) TO ADD THE MASTER MIX TO ONE SET OF +/+, -/-, AND +/- CONTROLS. Each shared gel will have only one set of 3 controls.
  8. Refer to the Bio-Rad Student Manual and the MEDBEd/SEEDBEd “PV92 PCR Student Instructions and Checklist” document for the Lesson #2 lab procedures for students.
  9. Follow the instructions listed on the “PV92 PCR Instruction Summary for Bio-Rad MyCycler THERMAL CYCLER” document provided by MEDBEd. The PCR amplification process will take almost 3 hours to complete. However, the machine can be left running overnight. When the process is complete, the machine will refrigerate the samples until you retrieve them the next day from the thermal cycler.

_____ 12. Final set upfor Day #3 or Lesson #3 Lab:

  1. Remove the PCR tubes from the thermal cycler and store in refrigerator until ready for Lesson #3.
  2. From the freezer, get the vial of Molecular Mass Ruler and allow it to thaw briefly. Label 6 tubes “MMR” and aliquot 11 µL of molecular mass ruler into each of the 6 MMR tubes. Refrigerate until ready for Lesson #3. Refreeze the remainder of the unused MMR in its original vial.
  3. From the refrigerator, get the vial of XC loading dye. Label 6 tubes “LD” and aliquot 90 µL into each tube. Please note that this 100 µL should be plenty for 5 students (10 µL each) and for the 3 controls (10 µL each). Later, during Lesson #3, remind one team per shared gel, to add loading dye to the controls. (The MMR already has a loading dye in it.)
  4. Set up 6 student workstations with the equipment listed on the Student Workstation Inventory for Day #3.
  5. The mini centrifuge machines provided by MEDBEd have adaptors to be used with the PCR tubes. Instead, you may also use capless micro test tubes as adaptors, if you wish. When ready to begin Lesson #3, students will need to centrifuge their PCR tubes for about 3 seconds.
  6. For a review of how to place gels into the electrophoresis chamber, a review of how to use the micropipets, how to stain the gels, and much more, see the MEDBEd/SEEDBEd “PV92 PCR Student Instructions and Checklist” document for Lesson #3.
  7. Refer to the “PV92 PCR Teacher’s Instruction Summary for Bio-Rad PowerPac Basic Power Supply and Bio-Rad Wide Mini-Sub Cell GT Electrophoresis Chamber” document for information on how to use the power supply.
  8. Refer to the Bio-Rad Student Manual and the MEDBEd/SEEDBEd “Student Instructions and Checklist” document for the Lesson #3 lab procedures for students.

_____ 13. For Lesson #4 and #5 dealing with analysis and interpretation of results and data, see the Bio-Rad Biotechnology Explorer Chromosome 16: PV92 PCR Informatics Kit Instructor’s Manual.

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