PV92 PCR
Student Instructions and Checklist
(notes from Donita and Donna 3-10-11)
This document includes detailed student instructions for the PV92 PCR lab in a checklist
format and also includes instructions on:
· how to make agarose gels
· how to store gels
· how to place the gels into the electrophoresis chamber
· how to use the mini centrifuge
· how to use micropipets
· how to stain and destain gels
IMPORTANT! Safety of students is the teacher’s responsibility. Prior to using this
instruction checklist, the power supply, and the electrophoresis chamber with students, the
teacher should be familiar with the:
· the Bio-Rad Biotechnology Explorer PV92 PCR Instruction Manual given to teachers at the SEEDBEd/MEDBEd Workshop for High School Teachers,
· the Bio-Rad safety precautions for the Bio-Rad PowerPac Basic Power Supply found at http://www.bio-rad.com/cmc_upload/Literature/51585/4006213E.pdf and in the manual packed with your Bio-Rad equipment
· the Bio-Rad safety precautions for the Bio-Rad Wide Mini-Sub Cell GT Electrophoresis Chamber found at http://www.bio-rad.com/cmc_upload/Literature/38717/M1704400B.PDF and in the manual packed with your Bio-Rad equipment
· the Bio-Rad safety precautions for the Bio-Rad MyCycler Thermal Cycler found at http://www.bio-rad.com/cmc_upload/Literature/52004/4006219F.pdf
· the Bio-Rad safety precautions for the Bio-Rad Temperature Controlled Water Bath and in the manual packed with your Bio-Rad equipment
Team members: ______
PV92 PCR LAB INSTRUCTIONS...CHECKLIST FOR STUDENTS
MAKING THE AGAROSE GEL (Gels may be prepared at any time prior to Lesson #3)
_____ Please skip this page (#2) and the next page (#3) if the gels have already been prepared for you.
_____ Wear gloves, goggles, & lab coat/apron if available.
_____ At the top of this page, list the names of all students in your team.
_____ If a Workstation Inventory form is available, confirm that all of the listed supplies are in your work area now and at the end of lab. Notify instructor if something is missing.
_____ Clean the chemical scoop with a paper towel dampened with 70% alcohol.
_____ Secure 1 piece of weighing paper, the agarose powder, and access to a balance.
_____ Place weighing paper on the balance pan and “zero the balance.” With the clean dry scoop, measure out .70 grams of agarose powder onto the weighing paper. ( .7 grams of agarose is about ¼ level teaspoon.)
_____ Pour the agarose powder from the weighing paper into a 125 ml Erlenmeyer flask
_____ Measure 70 ml of 1x TAE solution in a 100 ml graduated cylinder. Place the cylinder on a level surface and read the bottom of the meniscus at eye level.
_____ Add the 70 ml of TAE to your agarose powder in the flask and swirl the liquid in the flask for about 10 seconds
_____ Secure some hot pads and then heat the agarose/TAE solution for 30 seconds in the microwave oven.
_____ Using a hot pad, remove the solution from oven and swirl for about 10 seconds.
_____ Again, heat the solution for 30 seconds in the microwave. Using hot pad, remove the flask from oven and swirl for another 10 seconds
_____ Check to see if the agarose powder is all dissolved and gel solution is transparent. If the solution is not clear, heat the solution for 15 seconds, swirl and recheck. Repeat this step if necessary. Please note that the solution should momentarily reach a slow boil and then be immediately removed from the microwave oven. Avoid rapid boiling because steam will escape quickly and the agarose solution will become too concentrated.
_____ When the solution is completely clear, use your hot pads to carry the flask to your lab station where you will allow it to cool at room temperature for approximately 12 minutes to about 60˚C. Record the starting time of the cooling period. While you are waiting for the solution to cool, continue to follow the steps on this checklist.
_____ Rinse the scoop with water at the sink. Clean the scoop with a paper towel dampened with alcohol. Dry it and put it away. Wash your graduated cylinder, dry it, and put it away.
_____ Secure a leveling bubble, gel tray, gel comb, two dams and one electrophoresis chamber. With a paper towel dampened with 70% alcohol solution, wipe the surfaces of these items.
_____ Insert the gel tray and dams into the electrophoresis chamber as shown by your instructor. Do not add the solution yet.
_____ Place the leveling bubble in the tray. If the air bubble is not centered in the window inform your instructor. A level tray will produce a gel with uniform thickness.
_____ Fit the gel comb handle into the 2 gel tray slots that are about 1 cm from the dam nearest the black electrode end of the chamber.
_____ IMPORTANT!! Ask your instructor to check your gel tray installation, the leveling bubble, the comb installation, and the temperature of your gel solution before proceeding to the next step. (If your solution has cooled too much and has turned into a cloudy gel, you will need to reheat the gel until it becomes a clear liquid again. Then allow the reheated solution to cool to about 60˚C.)
_____ Return the leveling bubble to its proper storage location.
_____ After securing instructor approval, pour the gel solution into the gel tray. With a clean pipet tip, “pop” any bubbles or “pull” them to the edges of the gel. Record the starting time and allow to cool for about 30 minutes or until firm. Do not move the solution while it is cooling. The solution will become cloudy as it becomes a gel.
_____ While the solution is turning into a gel, wash the flask and put it away.
_____ When the gel is firm and has cooled to room temperature, remove the comb from the gel by slowly rocking the comb and pulling upward slowly. Rinse, dry and return comb to the storage area.
_____ Carefully remove the dams, casting gates, from the chamber so as not to tear or break the gel. Rinse, dry, and return dams to the storage area. Skip the next 3 steps if you will be using the agarose gel immediately.
_____ If you wish to store the gel in the gel tray and in the electrophoresis chamber until the next day, completely fill the reservoirs and cover the gel with TAE solution to keep the gel from drying out. This may sit at room temperature overnight. Skip the next 2 steps.
_____ If you wish to store the gel, overnight, in the gel tray but removed from the electrophoresis chamber, secure a resealable gallon size plastic bag and a permanent marker. Label the plastic bag with your name and date. Add about 50 ml of TAE to the bag to keep the gel moist. Store tray with gel and TAE in the bag at room temperature or in the refrigerator. Skip the next step.
_____ If you wish to store the gel for 2 to 7 days, secure a resealable plastic bag and a permanent marker. Label the plastic bag with your name and date. Slowly and carefully slide the gel out of your gel tray into your plastic bag. CAUTION: the fragile gel will break and tear if you are not careful. Add about 50ml of TAE solution to the bag to keep the gel moist. Store in the refrigerator. Rinse the gel tray with water and clean it with a paper towel dampened with 70% alcohol solution. Return tray to the storage area.
_____ Throw away your trash. Clean and put away all lab equipment. Use alcohol to wipe down the surface of your lab station.
_____ If you are working with a Workstation Inventory form, check it now and notify instructor if something is missing. If you are finished with this lab for today, give your Workstation Inventory to your instructor at this time.
Team members______
PV92 PCR LAB INSTRUCTIONS...CHECKLIST FOR STUDENTS
LESSON #1: DNA TEMPLATE PREPARATION
_____ Wear goggles, gloves, and lab coat/apron.
_____ At the top of this page, list the names of all students in your team.
_____ If a Workstation Inventory form is available, designate one person in your team to confirm that all of the listed supplies are in your work area now and at the end of lab. Notify instructor if something is missing.
_____ For each team member, obtain: (a) one screwcap tube containing 200µl InstaGene matrix,
(b) one empty 1.5 ml micro test tube with snap cap,
(c) one small paper cup containing approximately 10 ml saline solution.
_____ With a permanent marker, label each test tube and cup with your name or initials.
_____ Pour the saline solution from the cup into your mouth. Rinse vigorously for 30 seconds. Spit the saline solution back into the cup.
_____ Use the markings on the side of your snap cap test tube as a measurement guide and pour approximately 1 ml of your oral rinse into that test tube. Do NOT throw away the rest of your oral rinse. Close the snap cap.
_____ When all of your team is ready, take your snap cap micro test tubes to the mini centrifuge machine. As you load the centrifuge, make sure the hinges of the snap top tubes are all positioned farthest away from the center of the micro centrifuge machine. It is important that the test tube load should be completely balanced before spinning the micro centrifuge. Thus, completely fill all of the slots with test tubes or you may load the machine with only 2 or 4 tubes as long as the tubes are arranged symmetrically. IMPORTANT: Get your instructor’s approval before you turn on the micro centrifuge machine.
_____ Spin your tubes for 2 minutes. Keep the centrifuge lid closed until the rotor stops and then remove your test tubes. You should be able to see a pellet of whitish cells at the bottom of your tube. The pellet needs to be about the same size as a match head. If the pellet is too small, pour off the saline supernatant (the liquid above the cells) and add more of your own oral rinse and repeat the last 2 steps (above).
_____ Ask your instructor to check the size of your pellet before you proceed.
_____ Pour off and discard the supernatant. (A very small amount of supernatant will remain in the test tube.) You may also discard the remainder of your oral rinse now.
_____ Take the closed micro test tube containing your pellet to the vortexer machine. Hold the micro test tube while pressing it down onto the top of the vortexer. Vortex the pellet until no cell clumps remain and the cells are resuspended.
_____ Review this procedure for using the adjustable micropipets:
a. Do not lay the micropipet on the table. When not in use return it to the micropipet stand.
b. Adjust the volume dial if needed.
c. Attach a clean pipet tip to your micropipette as shown by your instructor. Do not touch the pipet tip.
d. In a sitting position, with both elbows on the table, hold the micropipet in a vertical position while in use.
e. Press the micropipet plunger to the FIRST stop.
f. Insert the pipet tip into the solution to be transferred.
g. SLOWLY release the plunger to retrieve the liquid. If you press the plunger beyond the first stop, you will pick up a larger volume of liquid and your measurement will be inaccurate.
h. Insert the pipet tip into the desired destination tube.
i. Press the plunger past the first stop to the SECOND stop to transfer the liquid.
j. Keep the plunger pressed when lifting the pipet tip out of the tube.
k. Eject the used pipet tip into the trash container. Use a clean tip for each transfer.
_____ Set your micropipet to 20µL and transfer all of your cheek cells into your screwcap tube containing the InstaGene. You may need to use the micropipette several times to transfer all of your cells.
_____ Screw the caps tightly on the tubes. Vortex the test tubes to mix the contents.
_____ When all of your team members are at this point, load the screw cap tubes into a foam micro test-tube holder. The bottom of the tubes should hang slightly below the foam holder. Float the holder with tubes in a 56°C water bath for 5 minutes. Record your starting time.
_____ Remove your foam holder from the 56°C water bath and vortex each of the test tubes several times.
_____ Return the tubes to the foam holder and place in the 56° C water bath for an additional 5 minutes.
_____ Remove your foam holder from the 56°C water bath and vortex each of the test tubes several times.
_____ Return the tubes to the foam holder and place in the 100° C water bath for 5 minutes.
_____ Remove your foam holder from the 100°C water bath and vortex each of the test tubes several times to resuspend the sample.
_____ When all of your team members are ready, place your test tubes in a balanced arrangement in the mini centrifuge machine. IMPORTANT: Get your instructor’s approval before you turn on the micro centrifuge machine.
_____ Centrifuge the test tubes for 5 minutes at 6,000xg or 10 minutes at 2,000xg.