BME100 s2015:Group6 12pmL4

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Contents

OUR TEAM

Name: Justin Blommer
Name: Justin Blommer
Name: Ethan Mathew
Name: Ethan Mathew
Name: Spencer Cobb
Name: Spencer Cobb
Name: Sarah Jones
Name: Sarah Jones
Name: Abigail Rene
Name: Abigail Rene

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP's
  • DNA/primer mix, 8 tubes, 50μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer
  • A strip of empty PCR tubes
  • Disposable pipette tips: only use each only once. Never re-use disposable pipette tips or samples will be cross-contaminated
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups


PCR Reaction Sample List

Assigned Tube Label Actual Tube Label PCR Reaction Sample Patient ID
G6 P 1 Positive control none
G6 N 2 Negative control none
G6 1-1 3 Patient 1, replicate 1 11105
G6 1-2 4 Patient 1, replicate 2 11105
G6 1-3 5 Patient 1, replicate 3 11105
G6 2-1 6 Patient 2, replicate 1 22923
G6 2-2 7 Patient 2, replicate 2 22923
G6 2-3 8 Patient 2, replicate 3 22923


DNA Sample Set-up Procedure

Step 1: Gather all materials: A PCR tube rack, 8 PCR tubes of 50 μL of PCR mix, 1 PCR tube of positive control, 1 PCR tube of negative control, 3 PCR tubes of Patient 1 DNA, 3 PCR tubes of Patient 2 DNA, PCR tube rack, micropipettor, micropipette tips, plastic cup, lab coat and gloves, and an OpenPCR machine.

Step 2: Label the sides of the tubes with Group 6's modified tube labels and place them in the rack.

Step 3: The 8 PCR tubes already containing the PCR reaction mix will be used in the the PCR machine. The provided DNA primer mixes will be pipetted into these 8 PCR reaction mix tubes.

Step 4: Transfer 50 μL of positive control into tube #1.

Step 5: Transfer 50 μL of negative control into tube #2.

Step 6: Transfer 50 μL of Patient 1 (ID: 11105) DNA sample into tubes #3, #4, and #5.

Step 7: Transfer 50 μL of Patient 2 (ID: 22923) DNA sample into tubes #6, #7, and #8.

Step 8: Now, there are 100 μL of complete PCR reaction in each tube.

Step 8: Ensure the lids are closed tight. Place the tubes in the PCR machine’s heating block and once all slots in the machine are filled, start the machine cycle.

Note: Ensure to replace pipette tip with a clean one between every pipette.


OpenPCR program

image:opew.jpg




Research and Development

PCR - The Underlying Technology

Function of PCR Reaction Components

The primary components of a polymerase chain reaction (PCR) include template DNA, primers, taq polymerase, and deoxyribonucleotides (dNTP's). They each serve a unique and equally important function in the PCR process. Template DNA contains the sequence of DNA that needs to be replicated and serves as a template for the making of the complementary strand. Primers are designed to match to the target segment of DNA to be copied, bind to a region on that DNA sequence and to start the DNA replications. Taq polymerase is thermostable DNA polymerase that adds complementary nucleotides onto the DNA strand. DNA polymerase continues until it gets to the end of the strand and falls off. Deoxyribonucleotides (dNTPS's) are dATP, dTTP, dCTP, and dGTP that build up DNA. They are used to create billions of DNA copies.


PCR Reaction Steps

The initial step of thermal cycling requires the DNA sample to be heated to 95°C for two minutes. This initial cycle at high temperature serves to unwind the DNA sequence in preparation for step two. Next, the DNA must be denatured at 95°C for thirty seconds resulting in two single-sided strands of DNA. The DNA must then anneal at 57°C for thirty seconds. In this step, the primers bind to their complimentary base pairs on the DNA, completely surrounding the target sequence to be copied. After annealing, the DNA must extend for thirty seconds at 72°C. The taq polymerase binds to the primer locations and extends the second strand of DNA until it has reached the end of the original target DNA strand.

This process of denaturing, annealing, and extending is repeated for a total of 35 cycles. This allows for production of millions of copies of the target DNA. The final step involves a two minute hold at 72°C. This extra time is to ensure that there are no single strands of DNA remaining. Lastly, a final hold at 4°C is done so the new DNA segments can be stored temporarily. The lower temperatures are used so that the new DNA segments do not begin to separate again.


Base Pairing

DNA consists of 4 different base pairs: Adenine, Thymine, Guanine, and Cytosine. They are commonly abbreviated as A, T, G, and C; respectively. Adenine and Thymine pair together, and Guanine and Cytosine pair together. Base pairing occurs during the annealing and extension steps of thermal cycling. In the annealing step, the mixture is cooled, allowing the primers to bind to their complimentary sequence in the template DNA. During extension, the DNA is heated, causing the DNA polymerase to extend the primers adding the nucleotides, using the target DNA as a template.

Source: http://www.uvm.edu/~cgep/Education/PCR.html


Bonus Digital PCR Process


image:rtrt1.jpg

  • Pictured above is the initial DNA strand before heat is applied
  • The initial cycle requires the temperature to increase to 95°C for 2 minutes

image:rtrt2.jpg

1) The temperature is increased to 95°C for 30 seconds. The strands of DNA separate into single strands

image:rtrt3.jpg

2) The temperature is decreased to 57°C and primers bind to complimentary matches on the target DNA sequence

image:rtrt4.jpg

image:rtrt5.jpg

3) The temperature is increased to 72°C and taq polymerase binds to the primers and add nucleotides to extend the second strand


  • Steps 1-3 are repeated for a total of 35 cycles

Source: http://www.dnalc.org/view/15475-The-cycles-of-the-polymerase-chain-reaction-PCR-3D-animation.html





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