BME100 f2014:Group8 L4

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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

Robert Schultz
Isabella Germinario
Bakir Mousa
Daniel Hersey
Ethan Lloyd
Bailey Myers

LAB 4 WRITE-UP

LAB A

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s(http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq-colorless-master-mix-m714-protocol/)
  • 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

PCRTableGroup8(2).png












OpenPCR Program Settings

The following values were used to run a heating & cooling program on the thermal cycler.

  • 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
  • Extend at 72°C for 30 seconds
  • FINAL STEP: 72°C for 2 minutes
  • FINAL HOLD: 4°C

Research and Development

Part 1- The Function of Each Component of a PCR reaction-

  • Template DNA- This provides a set of nucleotides for the primers to bond to and then to be replicated.
  • Primers-These sets of nucleotides bind to the template DNA and begin the replication process.
  • Taq Polymerase- This enzyme combines nucleotides to make a new strand of DNA which replicates the template strand
  • Deoxyribonucleotides (dNTP's)- These are the building blocks for each DNA strand. These are combined to form new strands of the selected DNA.

Part 2- The Steps of Thermal Cycling-

  • Initial step: 95°C for 3 minutes-
  • Denature at 95°C for 30 seconds-
  • Anneal at 57°C for 30 seconds-
  • Extend at 72°C for 30 seconds-
  • Final step: 72°C for 3 minutes-
  • Final hold: 4°C-

Part 3- DNA is made up of four types of molecules called nucleotides, designated as A, T, C and G. Base pairing, driven by hydrogen bonding, allows base pairs to stick together.

  • Adenine anneals to Thymine
  • Thymine anneals to Adenine
  • Cytosine anneals to Guanine
  • Guanine anneals to Cytosine

Part 4- Base pairing occurs during the Annealing and Primer Extension phases

LAB B