BME100 f2014:Group2 L4

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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: Victoria Bowman
Name: Victoria Bowman
Name: Andre Dang
Name: Andre Dang
Name: Chandler Heaton
Name: Chandler Heaton
Name: Jordan Shinn
Name: Jordan Shinn
Name: Abigail Weiss
Name: Abigail Weiss
Name: Alena Zapata
Name: Alena Zapata

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50 μL 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, each used only once
  • Cup for discarded tips
  • Micropipettor
  • Open PCR machine


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G2 + Positive control none
G2 - Negative control none
G2 1-1 Patient 1, replicate 1 13209
G2 1-2 Patient 1, replicate 2 13209
G2 1-3 Patient 1, replicate 3 13209
G2 2-1 Patient 2, replicate 1 26070
G2 2-2 Patient 2, replicate 2 26070
G2 2-3 Patient 2, replicate 3 26070


DNA Sample Set-up Procedure

  1. Step 1: Collect the positive and negative controls for the patient samples from the teaching assistant. Do no open the tubes.
  2. Step 2: Create labels that the group will use for the tubes. Our group, group two, used the tube labels G2+, G2-, G21-1, G21-2, G21-3, G22-1, G22-2, G22-3.
  3. Step 3: Record the patient ids in the Group 2 table.


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


Functions of the Components of PCR

There are several components of a PCR reaction that serve their own specific function. These components include template DNA, primers, taq polymerase, and deoxyribonucleotides. The function of extracted DNA from the cells is to serve as the template DNA from which the copies of DNA are made. This is important since the purpose of PCR is to make many DNA copies. The function of primers is to attach to a specific site of DNA in order to copy the desired DNA sequence. The primers have little chance of targeting the wrong site, so it is useful to successfully copy a desired DNA sequence. The function of the taq polymerase is to synthesise a new strand of DNA complementary to the starting template strand. The deoxyribonucleotides are the A’s, C’s, G’s, and T’s that make up DNA. Deoxyribonucleotides serve as the genetic building blocks that are used to create the DNA copies in a PCR reaction.

Steps of Thermal Cycling

During the initial step of thermal cycling which occurs at ninety five degrees Celsius for three minutes, the taq polymerase is being activated by the heat. It is heated to near boiling to allow the DNA to denature. During the denature step at ninety five degrees Celsius for thirty seconds, the double stranded DNA template denatures, so it is now single stranded DNA. Next, during the anneal step at fifty seven degrees Celsius for thirty seconds, short DNA sequences known as primers bind to, or anneal to, complementary matches on the single stranded DNA template. During the extend step at seventy two degrees Celsius for thirty seconds, the taq polymerase performs synthesis, it attaches nucleotides to the correct place along the single stranded DNA template so that a new complementary strand of DNA is extended from the primer. During the final step at seventy two degrees Celsius for three minutes, the taq polymerase ensures that any remaining single-stranded DNA is fully extended. During the final hold, there is a short term storage of the reaction.

Base Pairing and Thermal Cycling

The base adenine anneals to the base thymine. The base themine anneals to the base adenine. The base cytosine anneals to the base guanine. The base guanine anneals to the base cytosine. Base pairing occurs during two steps of thermal cycling, anneal and extend. During anneal, primers bind to complementary matches on the single stranded DNA template. During the extend step, the taq polymerase performs synthesis. The taq polymerase attaches nucleotides to the correct place along the single stranded DNA template to form a new complementary strand of DNA.

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