Human DNA Alu Amplification by Polymerase Chain Reaction (PCR)* Laboratory Procedure

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Clyde Stevenson
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1 Human DNA Alu Amplification by Polymerase Chain Reaction (PCR)* Laboratory Procedure *Polymerase Chain Reaction is covered by patents owned by Hoffmann-La Roche, Inc. This experiment was adapted from Laboratory DNA Science: An Introduction to Recombinant DNA Technology and Methods of Genome Analysis by Mark V. Bloom, Greg A. Freyer and David Micklos, copyrighted 1994, DNA Learning Center, Cold Springs Harbor Laboratory. 1

Amp Up Your DNA Polymerase Chain Reaction (PCR) Part I: DNA Isolation by Saline Mouthwash Step 1: Pour 10 ml of the saline solution (0.9% NaCl) into mouth and vigorously swish for 30 seconds.

2 Amp Up Your DNA Polymerase Chain Reaction (PCR) Part I: DNA Isolation by Saline Mouthwash Step 1: Pour 10 ml of the saline solution (0.9% NaCl) into mouth and vigorously swish for 30 seconds. Step 2: Expel saline solution into a paper cup. Step 3: Swirl to mix cells in the cup and transfer solution back into your 15 ml tube and cap tightly. Label the 15 ml tube with your initials. Step 4: Place your sample tube, together with other student samples, in a balanced configuration in a clinical centrifuge, and spin for 10 minutes at 2000 rpm. STOP and WAIT for your instructor to demonstrate the use of the micropipette. 2

Step 5: Carefully pour off supernatant into paper cup or sink. Be careful not to disturb the cell pellet at the bottom of the test tube. A small amount of saline will remain in the tube.

(If needed, 30 µl of saline solution may be added to facilitate resupension.) Step 7: Withdraw 30 µl of cell suspension and add to tube containing 100 µl of Chelex. Tap tube to mix.

3 Step 5: Carefully pour off supernatant into paper cup or sink. Be careful not to disturb the cell pellet at the bottom of the test tube. A small amount of saline will remain in the tube. Pour until level is 2mL. Step 6: Resuspend cells in remaining saline by sharply tapping tube on lab bench and pipetting in and out using the p200 micropipette. (If needed, 30 µl of saline solution may be added to facilitate resupension.) Step 7: Withdraw 30 µl of cell suspension and add to tube containing 100 µl of Chelex. Tap tube to mix. Label tube with your initials. Step 8: Bring your tube to the instructor to place in the boiling water bath for 10 minutes. Then cool briefly on ice or in the freezer. Step 9: After cooling, shake tube. Place in a balanced configuration in a microcentrifuge, and spin for 2 minutes. Stop: Store your sample on ice or in the freezer until ready to begin Part II. 3

Part II: DNA Amplification by PCR Step 1: Use a micropipette with a fresh tip to add 22 µl of PCR mix (containing primer and loading buffer) to a PCR tube containing a Ready-To-Go PCR Bead.

4 Part II: DNA Amplification by PCR Step 1: Use a micropipette with a fresh tip to add 22 µl of PCR mix (containing primer and loading buffer) to a PCR tube containing a Ready-To-Go PCR Bead. Tap tube lightly on bench or with finger to dissolve bead. Step 2: Using a fresh tip add 3 µl of your DNA (from Part I, step 9) to the reaction tube. Pipette from the supernatant and avoid cell debris and Chelex beads at the bottom. Tap tube lightly on bench or with finger to mix reagents. Do NOT label your small reaction tube. Stop and Wait for your instructor to explain the function of a thermocycler. Step 3: The instructor will keep track of where each students tube is loaded into the thermocycler. Again, do NOT label your reaction tube. Step 4: Once everyone s tubes are in the thermocycler they will be cycled automatically by the following program. Denature at 94 C for 30 seconds Anneal at 58 C for 30 seconds Extend at 72 C for 30 seconds 4

Part III: DNA Analysis by Gel Electrophoresis One gel will be made for every 2 people.

The agarose powder is located next to the balances. The electrophoresis buffer solution is in a bottle at your station.

Step 1: Write the number of your test tube on your data sheet.

5 Part III: DNA Analysis by Gel Electrophoresis One gel will be made for every 2 people. At your station you will find one numbered test tube, which will be used to mix the agarose powder and the buffer solution. The agarose powder is located next to the balances. The electrophoresis buffer solution is in a bottle at your station. Wait for the instructor to demonstrate the use of the equipment. Then follow the procedure below. Step 1: Write the number of your test tube on your data sheet. Step 2: Bring the numbered test tube(s) and beaker at your station to the electronic balance. Use the electronic balance to measure 0.3 grams of agarose into your test tube. Step 3: Return to your station. Use the 10 ml pipette to add 15 ml of electrophoresis buffer to the agarose powder in your test tube. 5

Step 4: Use the vortex to gently mix the agarose-buffer solution (speed=3). Mix carefully so that the agarose does not spill. Step 5: Bring the tube to the instructor who will add 1.

6 Step 4: Use the vortex to gently mix the agarose-buffer solution (speed=3). Mix carefully so that the agarose does not spill. Step 5: Bring the tube to the instructor who will add 1.5 µl SYBR Safe DNA Gel Stain to the agarose-buffer solution. The instructor will put the test tube with the agarosebuffer/sybr Safe stain solution into a boiling water bath. The solution will remain in the water bath until the agarose is completely dissolved (about 5 minutes). The instructor will notify you when the solution is ready. Stop/Stop/Stop/Stop/Stop/Stop/Stop/Stop/Stop Wait for the instructor to demonstrate pouring the gel and cleaning the test tubes. Step 6: Place the casting tray into the casting deck. The "casting deck" acts as the mold into which you will pour the gel. The "casting tray" is the glass slide. casting tray Step 7: casting deck Place the casting deck on a level surface. Step 8: When your number is called, bring your empty beaker to the instructor to retrieve your test tube. CAUTION: The test tube is hot. Keep the test tube in the beaker until it cools sufficiently to touch it (about 2-3 minutes). 6

7 Step 9: When the test tube is cool enough to touch, pour the agarose into one side of the casting deck. agarose Step 10: casting deck Insert a comb into the slots at the end of the casting deck. Make sure the long side of the comb faces the center of the casting deck. long side comb Step 11: casting deck Wash the test tube several times using the scrub brush. Rinse the test tube 10 times with water. Hang the test tube on the drying rack above the sink. Step 12: Wait for the gel to harden. It will become translucent. Do not touch the gel without gloves. Stop/Stop/Stop/Stop/Stop/Stop/Stop/Stop/Stop Wait for the instructor to demonstrate the removal of the gel and preparation of the electrophoresis box. 7

8 Removal of the gel *put on a pair of gloves* Step 13: Remove the comb from the gel. The depressions left in the gel are referred to as "wells". Step 14: Score all four sides of your gel with the flat end of your spatula. Step 15: Put your thumb through the hole in the bottom of the casting deck and gently push on the casting tray (the glass slide) while wiggling your thumb until the gel comes out. (If you have difficulty, ask an instructor for assistance.) Step 16: Keep the gel on the casting tray and place it on the central platform of the electrophoresis box so that the "wells" are at the NEGATIVE (black electrode) end. note: two gels will fit in each electrophoresis box, side by side. electrode end side view of loaded gels wells electrode end top view of loaded gels electrophoresis box platform gel gels 8

9 Step 17: Slowly pour the contents of the bottle labeled Electrophoresis Buffer" into the electrophoresis box. Fill the electrophoresis box until the gels are covered with a 2-3 mm layer of buffer. You can pour the buffer directly from the bottle. buffer 2-3 mm gel buffer level Stop/Stop/Stop/Stop/Stop/Stop/Stop/Stop Wait for the instructor to demonstrate preparing the cut DNA for electrophoresis. 9

10 Load the PCR products into the wells Note: Keep a record of the well you load on your data observation sheet. Be sure to practice loading gels before you load your DNA samples in the wells. Technique for loading gels: well pipette tip buffer level well pipette tip correct way to insert the pipette into well incorrect way to insert the pipette into well Step 18: Load 8-10µL of the DNA Standard into the middle well. Keep a record on your data sheet of the well you loaded. Step 19: Load 20µL of your PCR reaction into a well. Keep a record on your data sheet of the well you loaded. Step 20: CAUTION: HIGH VOLTAGE Gently place lid on electrophoresis box to avoid exposure to high voltage. Make sure metal electrodes are properly through slots on lid. The lid should be very loose on the chamber. Stop/Stop/Stop/Stop/Stop/Stop/Stop/Stop Wait for an instructor to demonstrate or assist you individually before connecting the cables and turning the power on. 10

11 Run the Electrophoresis Step 21: Connect the red cable to the red plug on the power source. Step 22: Connect the black cable to the black plug on the power source. Step 23: Press the switch to turn on the power source and set it to 75 volts. Step 24: You should see a distinct stream of bubbles floating up from the bottom of the chamber on the right side. IF YOU DO NOT SEE A STREAM OF BUBBLES, THE ELECTROPHORESIS IS NOT WORKING. Seek assistance from the instructor. Step 25: Keep the power on until the loading dye in each sample reaches at least half way across the gel. Do not let the dye run off the gel. Stop/Stop/Stop/Stop/Stop/Stop/Stop/Stop Wait for an instructor to demonstrate safely turning off the electricity and removal of the gels from the electrophoresis box. 11

12 Removal of the gel from the electrophoresis box Step 1: TURN THE POWER OFF. Step 2: DISCONNECT THE CABLES from the power source. Step 3: Gently lift the lid straight up off the electrophoresis chamber. Wrap cords and return the lid to the shelf. Part IV: Analysis of the gel Step 4: *wear gloves when handling the gel* Gently lift the casting tray (glass slide) and gel out of the box and place it on a plastic dish. Step 5: Bring the gel to the instructor. The DNA will be visualized by placing the gel on a Safe Imager TM light source. Step 6: *wear safety glasses when using the Safe Imager TM * Observe the banding patterns on your gel and sketch the results on your data sheet. 12

13 Analysis of the gel: 1) Examine the gel containing your sample and those from other individuals. Orient the gel with the sample wells at the top. First, ascertain whether or not you can see a diffuse (fuzzy) band of "primer dimer," that appears at the same position in each lane toward the bottom of the gel. Primer dimer is not amplified human DNA, but is an artifact of the PCR reaction that results from primers amplifying themselves. Excluding a primer dimer, interpret the allele bands in each lane of the gel which appear close to the 400 and 700 bp DNA standard bands. a) No bands visible. This usually results from an error during sample preparation, such as losing the cheek cell pellet or failing to resuspend Chelex beads prior to transferring solutions between test tubes. b) One band visible. The simplest explanation is that the individual is homozygous either for the Alu insertion or for the absence of the Alu insertion. c) Two bands visible. The individual is heterozygous for the Alu insertion. d) Three or more bands visible. The two brightest bands are likely the true alleles. Additional bands may occur when the primers bind nonspecifically to chromosome loci other than Alu and give rise to additional amplification products. 2) After observing the banding patterns on your gel, draw a picture of your results on your data observation sheet. Note: In the lane with the standard, you should see bands of DNA with the following base pair sizes: 13

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