BME100 s2015:Group18 12pmL4

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Contents

OUR TEAM

Name: Walter C. BregonRole(s): Chief of Neurosurgery
Name: Walter C. Bregon
Role(s): Chief of Neurosurgery
Name: Jenna TarasRole(s)
Name: Jenna Taras
Role(s)
Name: EyerusalemRole(s)
Name: Eyerusalem
Role(s)
Name: Nathan LeFortRole(s): being superior
Name: Nathan LeFort
Role(s): being superior
Name: Abdurrahman DarwishRole(s)
Name: Abdurrahman Darwish
Role(s)
Name: Hau NguyenRole(s):Research & Development
Name: Hau Nguyen
Role(s):Research & Development

LAB 4 WRITE-UP

Protocol

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/gotaqcolorless-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

Tube Label PCR Reaction Sample Patient ID
G18 P Positive control none
G18 N Negative control none
G18 1-1 Patient 1, replicate 1 41180
G18 1-2 Patient 1, replicate 2 41180
G18 1-3 Patient 1, replicate 3 41180
G18 2-1 Patient 2, replicate 1 68784
G18 2-2 Patient 2, replicate 2 68784
G18 2-3 Patient 2, replicate 3 68784


DNA Sample Set-up Procedure

  1. Collect a small sample of DNA from cells such as blood, skin, saliva or even hair follicles
  2. Move your extracted DNA sample into a special PCR tube
  3. Add primer 1 to the PCR tube (Primer 1 attaches to a site on the DNA strand at either end of the template segment)
  4. Add primer 2 (which attaches to the 2nd site of the template segment on the DNA strand) to the PCR tube
  5. Add nucleotides to the PCR tube
  6. Add DNA polymerase to the PCR tube
  7. Now place the PCR tube containing all of the reaction components into the DNA thermal cycler
  8. Push start button on DNA thermal cycler


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

Q1. What is the function of each component of a PCR reaction?

Image:L4-T1.png

Q2. What happens to the components (listed above) during each step of thermal cooling?

Image:L4-T2.png

Q3. 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. Which base anneals to each base listed below?

Adenine-Thymine, Thymine-Adenine, Cytosine-Guanine, Guanine-Cytosine



Bonus Round

Image:Poor_PCR.png

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