Intro
The blueprint that controls a large portion of our appearance, behavior, and tendencies is called our Genome. The gonome is composed of DNA and is our hereditary code. One method of expanding and cpying our genome is called PCR, or Polymerase Chain Reaction. Essentially, PCR allows us to produce larege quatities of DNA in a test tube , starting from only a trace ammount. Trace ammounts can be anything from a drop of blood, a hair follicle, or even a cheek cell. PCR has left an imprint on four main areas of genomic research: gene mapping, cloning, DNA sequencing, and gene detection. To use DNA for PCR, you have to extract living cells from your body. Once done, you place desired cells into a micro test tube conataining IntstaGene Matrix Beads. These beads are responsible for killing DNAse in the cell so that once the membrane in broken, the DNA isn't dammaged.
Procedure
DAY 1: Cheek Cell DNA Template Preparation
1. Label on 1.5 mL micro test tube with your initials. Label one screwcap tube containing 200 microliters of InstaGene matrix with your initials.
(Rinse Out Mouth if Necessary)
2. Obtain a cup containing saline solution from your instructor. Chew the insides of your cheeks gently, and pour the saline into your mouth and rinse vigorously for 30 seconds. Expel the saline back into the cup.
3. Transfer 1 mL of your saline rinse into the micro test tube (NOT the screwcap tube) with your initials.
(Save cup for step 5)
4. Spin your tube in a balanced centrifuge at full speed for two minutes. At the bottom of the tube you should see a match-head sized pellet of whitish cells at the bottom of the tube.
5. After pelleting cells, pour off the saline into your cup being careful not to lose your pellet.
6. Re-suspend pellet by flicking or vortexing the tube so no clumps remain.
7. Using a 2-20 micro-pipette, set it at 20 microliters and transfer all of your suspended cells into the screwcap labeled InstaGene. Discard the flip-top tube.
8. Screw the cap tightly on the tube and shake or vortex the tube to mix the contents.
9. When all members have collected their cells, place tubes in foam micro test-tube holder, and incubate at 56 degrees C for 10 minutes in a water bath.
10. Remove tubes, shake or vortex, and place the tubes in a boiling water bath (100 degrees C) for 5 minutes.
11. Remove tubes from boiling water bath and shake or vortex to re-suspend. Pellet the matrix by spinning at 6,000 x g for 5 minutes.
12. Store screwcap in the refrigerator until the next day.
DAY 2: PCR Amplification (Use PCR tips today)
1. Obtain tube with your DNA template from the refrigerator. Spin the screwcap tube for 2 minutes at 6,000 x g in a centrifuge.
2. Label PCR tubes with your initials and place PCR tube in your PCR rack.
3. Transfer 20 microliters of the DNA template from the screwcap into the bottom of the PCR tube. Be careful not to transfer any matrix beads into the PCR tube.
4. Put 20 microliters of Master Mix directly into the DNA template. Cap tube tightly. Mixture should be yellow.
5. Place PCR tube into the thermal cycler.
DAY 3: Electrophoresis of Amplified PCR Samples and Staining of Agarose Gels
1. Obtain PCR tube from the thermal cycler and place in the capless micro test tube. Pulse-spin the tube for approx. 3 seconds.
2. Add 10microliters of PV92 XC loading dye into your PCR tube and mix gently.
3. Place an agarose gel in the electrophoresis apparatus. Check that the wells of the agarose gels are near the black (-) electrode and the base of the gel is near the red (+) electrode.
4. Fill electrophoresis chamber and cover gel with 1x TAE buffer. This will require approx. 275 mL of 1x buffer.
5. Using a clean tip for each sample, load the samples into 8 wells in the following order:
Lane | Sample | Load Volume |
1 | MMR (DNA standard) | 10 microliters |
2 | Homozygous (+/+) control | 10 microliters |
3 | Homozygous (-/-) control | 10 microliters |
4 | Heterozygous (+/-) control | 10 microliters |
5 | Student 1 | 20 microliters |
6 | Student 2 | 20 microliters |
7 | Student 3 | 20 microliters |
8 | Student 4 | 20 microliters |
6. Secure the lid on the gel box. Red-Red, Black -Black. Connect electrical leads to power supply.
7. Turn on power and electrophorese your samples at 200V for 3 min.
Staining of Agarose Gel
When electrophoresis is complete, remove lid and place gel into tray.
DONE!!
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