PCR Lab
Classroom Day One
Updated Amgen Lab 8: Genomic DNA Extraction from Buccal Epithelial Cells and Amplification of the tPA Locus using the Polymerase Chain Reaction
Materials
Sterile pipette tips Chelex tube: 0.2 mL of 10% Chelex solution
Boiling water bath PCR tubes
High-speed centrifuge PCR Machine (Thermal Cycler) - college
Day ONE: Collecting your DNA sample for PCR thermocycler
1. Obtain a Chelex tube. Note that this tube is identified with a number. Record this number in your notebook. Only you will know this anonymous code.
2. To collect buccal epithelial cells, use a sterile toothpick or sterile pipette tip to gently scrape the inside of both cheeks. Scrape well so that you get lots of cells. However, this procedure should be non-invasive so don’t draw blood.
3. Transfer the cells that you have removed from the toothpick/pipette tip to the Chelex tube. Vigorously twirl the toothpick/tip in the Chelex resin to knock the cells off toothpick/ tip. This step is important; you want to get as many cells off the toothpick/pipette tip and into the Chelex tube as possible.
4. Close the Chelex tube tightly. Place your tube in the rack which will be placed in the boiling water bath or place your tube directly into the 100°C hot block. Boil or heat the cells for 10 minutes. This heating will lyse the cells and help to destroy some of the nucleases, which can degrade the DNA.
5. Use the high-speed centrifuge set to ~10,000 rpms to spin down the Chelex and cell debris for 5 minutes.
6. Using the P-20 pipette and a clean pipette tip, carefully remove 5 mL of supernatant (which contains your genomic DNA) and place it into a clean PCR tube. Avoid aspirating Chelex beads as this will inhibit the downstream PCR procedure. Label the cap and side of this PCR tube with your personal, anonymous code.
Classroom Day One
Updated Amgen Lab 8: Genomic DNA Extraction from Buccal Epithelial Cells and Amplification of the tPA Locus using the Polymerase Chain Reaction
Materials
Sterile pipette tips Chelex tube: 0.2 mL of 10% Chelex solution
Boiling water bath PCR tubes
High-speed centrifuge PCR Machine (Thermal Cycler) - college
Day ONE: Collecting your DNA sample for PCR thermocycler
1. Obtain a Chelex tube. Note that this tube is identified with a number. Record this number in your notebook. Only you will know this anonymous code.
2. To collect buccal epithelial cells, use a sterile toothpick or sterile pipette tip to gently scrape the inside of both cheeks. Scrape well so that you get lots of cells. However, this procedure should be non-invasive so don’t draw blood.
3. Transfer the cells that you have removed from the toothpick/pipette tip to the Chelex tube. Vigorously twirl the toothpick/tip in the Chelex resin to knock the cells off toothpick/ tip. This step is important; you want to get as many cells off the toothpick/pipette tip and into the Chelex tube as possible.
4. Close the Chelex tube tightly. Place your tube in the rack which will be placed in the boiling water bath or place your tube directly into the 100°C hot block. Boil or heat the cells for 10 minutes. This heating will lyse the cells and help to destroy some of the nucleases, which can degrade the DNA.
5. Use the high-speed centrifuge set to ~10,000 rpms to spin down the Chelex and cell debris for 5 minutes.
6. Using the P-20 pipette and a clean pipette tip, carefully remove 5 mL of supernatant (which contains your genomic DNA) and place it into a clean PCR tube. Avoid aspirating Chelex beads as this will inhibit the downstream PCR procedure. Label the cap and side of this PCR tube with your personal, anonymous code.
- Place your PCR tube and Chelex tube into the racks at the front of the room. Your genomic DNA sample can be stored in the freezer at -20ºC until your teacher is ready to run the PCR reaction.
PCR Lab
Classroom Day 2
At the college the Thermocycler:
o Your teacher will place 20 mL of Master Mix in your PCR tube. The Master Mix contains the two primers that target the tPA locus, dNTP’s (deoxynucleotide triphosphates: ATP, TTP, CTP and GTP), PCR buffer w/ MgCl2, molecular grade water (very pure) and Taq polymerase.
o It will take 2 ½ to 3 hours to complete the 30 PCR cycles.
o After the PCR run, your samples will be stored in the freezer.
Students Day TWO: Gel Electrophoresis and Photo
1. Obtain your numbered PCR tube containing your amplified DNA.
2. Add 4 mL of loading dye (Orange G) directly into your PCR tube.
3. 2% agarose gels have been prepared for your class and placed in gel electrophoresis chambers. 10 mL of a DNA ladder will be loaded into the first well of each gel.
4. Load 20 mL of your sample (now with loading dye) into one well of a gel. **Make sure you write down where you loaded your sample**
5. After every student has loaded their sample, connect the gel box to the power supply.
6. On the power supply, set the voltage to160 v and run the samples for approximately 20 minutes.
7. After the gel run is complete, your gels will be photographed by your teacher.
Classroom Day 2
At the college the Thermocycler:
o Your teacher will place 20 mL of Master Mix in your PCR tube. The Master Mix contains the two primers that target the tPA locus, dNTP’s (deoxynucleotide triphosphates: ATP, TTP, CTP and GTP), PCR buffer w/ MgCl2, molecular grade water (very pure) and Taq polymerase.
o It will take 2 ½ to 3 hours to complete the 30 PCR cycles.
o After the PCR run, your samples will be stored in the freezer.
Students Day TWO: Gel Electrophoresis and Photo
1. Obtain your numbered PCR tube containing your amplified DNA.
2. Add 4 mL of loading dye (Orange G) directly into your PCR tube.
3. 2% agarose gels have been prepared for your class and placed in gel electrophoresis chambers. 10 mL of a DNA ladder will be loaded into the first well of each gel.
4. Load 20 mL of your sample (now with loading dye) into one well of a gel. **Make sure you write down where you loaded your sample**
5. After every student has loaded their sample, connect the gel box to the power supply.
6. On the power supply, set the voltage to160 v and run the samples for approximately 20 minutes.
7. After the gel run is complete, your gels will be photographed by your teacher.
PCR Song
PCR = Polymerase Chain Reaction animation
The polymerase chain reaction (PCR) is widely used to amplify DNA regions of know sequenv=ces reaction mixture contains target DNA sequences to be amplified two primers heat-stable taq polymer nucleotides PCR starts WITH A DOUBLE-stranded DNA and each cycle of the reaction begins with a brief heat treatment to separate the two strands after strand separate cooling od the DNA is the presence of a large excess of the printer DNA this mixtures is then incubated with DNA polymerase. Taq polymerase adds nudeotides to the 3 ends of each printer the temperature is raised again to separate the DNA strands and then loaded suffuectly to allot the printers to attach as the procedures is performed over and over again the new synthesized fragments serve as template in the turn. in angles the proccess then repeats and copies the original DNA and then produce in a short time, the procedure is performed a=over and over again.