BME100 s2015:Group16 12pmL4

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Contents

OUR TEAM

Name: Hanna Monroe
Name: Hanna Monroe
Name: Natkanes Pakjirasirikul
Name: Natkanes Pakjirasirikul
Name: Trevaun Walker
Name: Trevaun Walker
Name: Reaghan Fletcher
Name: Reaghan Fletcher
Name:Thomas Murphy
Name:Thomas Murphy

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
  • 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
G16 + Positive control none
G16 - Negative control none
G16 1-1 Patient 1, replicate 1 47360
G16 1-2 Patient 1, replicate 2 47360
G16 1-3 Patient 1, replicate 3 47360
G16 2-1 Patient 2, replicate 1 31303
G16 2-2 Patient 2, replicate 2 31303
G16 2-3 Patient 2, replicate 3 31303


DNA Sample Set-up Procedure

  1. Label Tubes with the labels from the table above (e.g. G16 +, G16 2-2...).
  2. Transfer 50 μL of the + and - controls to the PCR tubes labeled G16 + and G16 - using the micropipette.
  3. Transfer 50 μL of the corresponding patient's DNA to special PCR tube using the micropipette.
  4. Dispose and replace the tips between each transfer.
  5. Place the PCR reaction tubes in the thermal cycler.


OpenPCR program

Image:OPPCR.jpg





Research and Development

PCR - The Underlying Technology

Question 1:

Image:g16questionone.png

Question 2:

Image:g16question2.png

Question 3:

Image:g16question3.png

Question 4:


Base pairing occurs in the anneal step and final step of the thermal cycling. In the anneal step at 57°C for 30 seconds, the primer is pairing with the DNA strand. In the final step at 72°C for 3 minutes, the DNA polymerase is paring nucleotides to complete the DNA strand.





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