BME100 f2014:Group30 L4

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Owwnotebook icon.png BME 100 Fall 2014 Home
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Name: Kyle Lindley
Name: Brittney Wong
Name: Calvin Baumgartner
Brody Senior Photo.jpg
Name: Robin Skaria
Name: Megan Wieser

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, 50 μL 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

Tube Label PCR Reaction Sample Patient ID
G30 + Positive control None
G30 - Negative control None
G30 1-1 Patient 1, replicate 1 56859
G30 1-2 Patient 1, replicate 2 56859
G30 1-3 Patient 1, replicate 3 56859
G30 2-1 Patient 2, replicate 1 50591
G30 2-2 Patient 2, replicate 2 50591
G30 2-3 Patient 2, replicate 3 50591


DNA Sample Set-up Procedure

  1. After heating and cooling the DNA sample, add Primer One followed by Primer Two to the sample with a micropipette.
  2. Add the nucleotides followed by the DNA polymerase to the DNA sample with the micropipette.
  3. Put the DNA Sample test tube into the thermal cycler and press start in order to complete the amplification process.


OpenPCR program

HEATED LID: 100°C

INITIAL STEP: 95°C for 2 minutes

NUMBER OF CYCLES: 35

Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 seconds

FINAL STEP: 72°C for 2 minutes

FINAL HOLD: 4°C






Research and Development

PCR - The Underlying Technology

Question 1: Functions of PCR Components:

The template DNA is the DNA sequence that is copied during the process of mRNA production. The base pairs of the template strand are then copied by DNA primers, as the primers attach to the desired DNA sequences to be copied. Taq polymerase binds to the DNA primers and adds corresponding nucleotides to the template strand in order to complete the second strand of the DNA. Since DNA is made up of deoxyribunucleotides, the deoxyribonucleotides are used to hold the double stranded DNA molecule together.

Question 2:

The initial step is to raise the temperature to near boiling. Causing the double-stranded DNA to separate, or denature, into single strands. When the temperature is decreased, DNA sequences known as primers bind or anneal to complementary matches on the target DNA sequence. The primers bracket the target sequence to be copied. At a slightly higher temperature the enzyme taqpolymerase binds to the prime sequences and adds nucleotides to extend the second strand. This completes the first cycle. In subsequent cycles, the process of denaturing, annealing, and extending are repeated to make additional DNA copies. After three cycles, the target sequence, defined by the primers, begins to accumulate. After 30 cycles, as many as a billion copies of the target sequence are produced from a single starting molecule.

Question 3: Corresponding Base Pairs:

The following nucleotide base pairs bind or anneal to each other during the process of of DNA replication during the PCR reaction.

Adenine (A) - Thymine (T); Thymine (T)-Adenine (A); Cytosine (C)- Guanine (G); Guanine (G)- Cytosine (C)