Download Slide 1 - Issaquah Connect

The Polymerase Chain Reaction
 The polymerase chain reaction, PCR, can produce
many copies of a specific target segment of DNA
 A three-step cycle brings about a chain reaction that
produces an exponentially growing population of
identical DNA molecules
 The key to PCR is an unusual, heat-stable DNA
polymerase called Taq polymerase.
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Figure 13.25
Technique
5
3
Target sequence
Genomic DNA 3
1 Denaturation
5
5
3
3
5
2 Annealing
Cycle 1
yields 2 molecules
Primers
3 Extension
New
nucleotides
Cycle 2
yields 4 molecules
Cycle 3
yields 8 molecules;
2 molecules
(in white boxes)
match target sequence
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Figure 13.25a
Genomic DNA
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5
3
3
Target sequence
(TAS2R38)
5
Figure 13.25b-1
1 Denaturation
Cycle 1
yields 2
molecules
© 2014 Pearson Education, Inc.
5
3
3
5
Figure 13.25b-2
1 Denaturation
5
3
3
5
2 Annealing
Cycle 1
yields 2
molecules
© 2014 Pearson Education, Inc.
Primers
Figure 13.25b-3
1 Denaturation
5
3
3
5
2 Annealing
Cycle 1
yields 2
molecules
Primers
3 Extension
New
nucleotides
© 2014 Pearson Education, Inc.
Figure 13.25c
Cycle 2
yields 4 molecules
Cycle 3
yields 8 molecules;
2 molecules
(in white boxes)
match target sequence
Results After 30 more cycles, over 1 billion (109) molecules
match the target sequence.
© 2014 Pearson Education, Inc.
© 2014 Pearson Education, Inc.
© 2014 Pearson Education, Inc.
So what are we doing with this technique???
We will use PCR + restriction digests to determine
your genotype for PTC tasting (TAS2R38)
Calendar
05.18.16
Isolate DNA from your cheek cells
05.20.16
Set up PCR using your DNA
05.24.16
Set up restriction digests of your PCR product
05.26.16
Run gels and analyze
What you’ll need to turn in on 06.01.16
1. Completed lab worksheet (5 pt)
2. Picture of your gel (5 pt)
3. Hand-drawn standard curve of your DNA marker (10 pt)
4. This will go into the main labs scoring (20 pt total)
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Day 1: Isolating your DNA
•Label two microtubes and a cup of saline solution with your identity number given to you by your teacher.
•Pour saline solution in your mouth and vigorously rinse your cheeks for 1 minute.
•Expel the saline solution back into the cup.
•Carefully, over the garbage or a sink, pour your spit and saline solution into your microtube.
•Close the microtube tightly and put it into the high powered microcentrifuge and spin it for 5 minutes at 10,000
rpm.
•Look for a cell pellet at the bottom of the tube and carefully pour the liquid above the pellet (called the
supernatant) into a waste container and keep the cell pellet in the tube.
•Pipette 500 λ of 10% chelex into your microtube and resuspend the cell pellet by gently mixing the solution up
and down in the pipette.
•Once resuspended fully, close the tube lid tightly and poke a hole in the top of the lid using a tack.
•Place the tube in the 99o hot block for 10 minutes.
•Resuspend the solution one more time and then spin in the high-speed centrifuge for 5 minutes so the Chelex
beads fall to the bottom of the tube.
•Without disturbing the beads at the bottom, pipette about 50 λ of the solution into second a tube labeled with
your name or identity number and the date. This is your extracted DNA. Your teacher will freeze your tube for
you to use in your next experiment.
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Day 2: Performing PCR
•Keep PCR tubes on ice. Label a PCR tube with your identity number and then put it back on
ice.
•Obtain your DNA tube from your teacher and carefully thaw it on the countertop.
•Pipette 5 λ of your DNA into your PCR tube.
•Come up to front of room with your 20 λ pipette set at 20 λ and with a fresh tip. Pipette 20 λ of
Master Mix from the teacher’s stock tube into your PCR tube.
•Return to your bench and quickly cap and vortex your PCR tube and then spin it down in the
low-speed minicentrifuge.
•Bring your PCR tube to the PCR machine and put your tube in the ice container (if your
teacher is waiting for other student tubes) or put your tube directly in the PCR machine.
•Your teacher will run the PCR machine once all tubes are in the PCR machine.
© 2014 Pearson Education, Inc.
Day 3: Restriction Digest of PCR Product
•Get your “D” microtube from your teacher and label it with your name. This tube has 2 λ of
HaeIII enzyme for the restriction digest.
•Label another microtube with your name and “U” for your undigested PCR product.
•Obtain your PCR product.
•Pipette 10 λ of PCR product into “U” tube.
•Pipette 12 λ of your PCR product into your “D” tube.
•Add 4 λ of 2.5 x Restriction Buffer to both the “U” and “D” tubes.
• Cap your tubes, mix them, and then spin them quickly in the microcentrifuge
•Put your “D” tube in the 37o C waterbath for 1 hour.
•Bring your “U” tube to your teacher who will pull your “D” tube out of the waterbath and put
both tubes into the refrigerator.
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Day 4: Analyzing your Restriction Digests using Agarose Gel Electrophoresis
•Obtain your “U” and “D” tubes.
•Set up your electrophoresis box with 1X TAE buffer and a 2% agarose gel. Be sure to
put the wells of the gel on the correct side of chamber (near the power supply).
•Pipette 2 λ of loading dye into your “U” tube and 2 λ of dye into your “D” tube.
•Load 10 λ of PCR Marker into the first well of your gel.
•Load 12 λ of “U” tube into the next lane.
•Load 12 λ of “D” tube into the next lane.
•Record which lanes your samples are in.
•When all lanes are loaded, close the electrophoresis box and run your gel at 125V for
35-45 minutes.
•Carefully remove your gel from the gel box when finished running.
•Take a picture of your gel using the LED camera.
•Analyze your results. Create a standard curve from the DNA ladder, then use it to
identify the weights of the different bands in your sample. Note your genotype based
on the band pattern.
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