Updated Amgen Lab 8: Genomic DNA Extraction from Buccal Epithelial Cells
and Amplification of the tPA Locus using the Polymerase Chain Reaction
MATERIALS
Equipment and supplies:
Sterile pipette tips
Boiling water bath
High-speed centrifuge
PCR tubes
PCR Machine (Thermal Cycler)
Reagents:
0.2 mL of 10% Chelex solution
Master Mix Reagents:
dNTP's (50mM)
Forward primer (4pm/uL)
Reverse primer (4pm/uL)
10X PCR buffer
Mgcl2
Taq Polymerase
Molecular grade water
METHODS
Day ONE: Collecting your DNA sample and running PCR
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 L 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.
7. 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.
8. Your teacher will place 20 L 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.
9. It will take 2 ½ to 3 hours to complete the 30 PCR cycles.
10. After the PCR run, your samples will be stored in the freezer.
Day TWO:
1. Obtain your numbered PCR tube containing your amplified DNA.
2.
Add 4 L 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 L of a DNA ladder will be loaded into the first
well of each gel.
4. Load 20 L 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.
Preparing samples and running PCR Teacher’s Guide:
PCR is a very sensitive process. The DNA samples can be easily contaminated.
Please wear gloves and perform all work carefully and as sterilely as possible!
Equipment and Supplies:
Thermal cycler
microfuge tubes
Permanent marker
Sterile toothpicks
Vortexer
Ice bucket/Ice
PCR tubes
Master Mix Reagents:
dNTP's (50mM)
Forward primer (4pm/uL)
Reverse primer (4pm/uL)
10X PCR buffer w MgCl2
Taq Polymerase
Molecular grade water
Remove reagents and DNA samples from the freezer and allow them to thaw on ice.
Leave Taq Polymerase in freezer until ready to use. Mix/Finger vortex all reagents just
prior to use.
Note: Change pipette tips after each use!
Prepare Master Mix in a clean (preferably sterile) eppendorf tube using volumes
calculated for total number of samples you're planning to run. Keep all reagents and
Master Mix on ice. Don't forget to add Taq Polymerase which is still in the freezer!
Add 20µL of Master Mix to each DNA sample for a total volume of 25uL (5uL DNA
sample + 20uL of Master Mix). Keep on ice.
Take samples to the PCR machine and Run program “Amgen PCR”. (2 ½ to 3 hours)
The last step of the PCR run will hold at 4 degrees Celsius for infinity so you do not have
to be present when the run is complete.
After the PCR run, place the samples in the freezer until the students are ready to run
their gels.
Next class period prepare gels, run samples, and take photos.
Master Mix for PCR Program for Amplifying
the ALU element (Amgen Lab 8)
PCR is a very sensitive process. The samples can be easily contaminated. Please
wear gloves and perform all work carefully and as sterilely as possible!
Vortex all reagents just prior to use and vortex the MM once all reagents are combined
Change pipette tips after each use!!!
Master Mix (MM) for 1 PCR
Sample:
Amount(s) needed for
___ samples
Molecular grade water 10.35uL
Forward primer (4pM/uL) 2.5uL
Reverse primer (4pM/uL) 2.5uL
dNTPs (10mM) 0.5uL
10x PCR buffer W/O Magnesium 2.5uL
25 mM Magnesium Chloride
1.5uL
Taq polymerase .15uL
Total Master Mix volume = 20uL
Total MM volume:
In each PCR tube 20 uL Master Mix + 5 uL Genomic DNA
for several samples multiply the amount of each reagent above by the
total number of samples
Total number of samples= # of student DNA samples + 4 extra to account for loss
during pipeting
July 2012