BME100 f2014:Group3 L4

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Contents

OUR TEAM

Name: Marissa Seelhammer
Name: Marissa Seelhammer
Name: Brianna Denuit
Name: Brianna Denuit
Name: Farhad Eghbalian
Name: Farhad Eghbalian
Name: Shane Mitchell
Name: Shane Mitchell
Name: Catherine Piatak
Name: Catherine Piatak
Name: Nivenka Mahesh
Name: Nivenka Mahesh

LAB 4 WRITE-UP

Protocol

Materials

  • Latex Gloves
  • Lab Coat
  • Micropipette
  • One empty strip of PCR tubes
  • PCR reaction mix
  • DNA/ primer mix
  • Disposable pipette tips
  • Cup for discarded tips
  • OpenPCR machine: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G# + Positive control none
G# - Negative control none
G# 1-1 Patient 1, replicate 1 37145
G# 1-2 Patient 1, replicate 2 37145
G# 1-3 Patient 1, replicate 3 37145
G# 2-1 Patient 2, replicate 1 19452
G# 2-2 Patient 2, replicate 2 19452
G# 2-3 Patient 2, replicate 3 19452

DNA Sample Set-up Procedure


1. Receive materials- Do not open the positive and negative controls. The positive control is DNA from someone who has tested positive for the disease. The negative control is DNA from someone who has teased negative for the disease.


2. Separate the empty strip of PCR tubes so it will fit into the OpenPCR machine.


3. Create labels for the PCR tubes in order to keep track of which tubes contain which patient's DNA.


4. Record patients' IDs.


5. With the 6 left over PCR tubes, micropipette 50 μL of PCR reaction mix into every tube.

Make sure to change the pipette tip after every use to avoid contamination.


6. Micropipette 50 μL of DNA from patient 1 into three out of the six PCR tubes from step 5.

Make sure to change the pipette tip after every use to avoid contamination.


7. Micropipette 50 μL of DNA from patient 2 into the other three PCR tubes from step 5 that DO NOT contain patient 1 DNA.

Make sure to change the pipette tip after every use to avoid contamination.


8. Close all of the PCR tubes securely.


9. Place the 6 tubes of solution that were mixed in steps 5-7 as well as the PCR tubes containing the positive and negative controls into two rows of the OpenPCR. Wait for group sharing the OpenPCR to place their tubes in before beginning the program.

OpenPCR program


Specifications for setting up the  openPCR device

Bonus

Image:Pcr med.jpg





Research and Development

PCR - The Underlying Technology

Template DNA is the original strand of DNA used to make copies. Primers are short pieces of DNA that are customized so they can have any sequence of nucleotides. 2 primers are made to match the sequence of DNA that is being copied. Taq Polymerase is an enzyme used in the PCR reaction that acts like a machine and matches the nucleotides with the DNA code making copies.

At 95 degrees celsius the DNA double helix is separated into 2 single stranded DNA molecules. It is then cooled down to 50 degrees celsius where the primers work their way in and lock onto their target strands before the strands can reconnect. It is then heated again to 72 degrees celsius where the DNA polymerase are activated and they locate a primer attached to a single DNA stand where it begins to add complementary nucleotides onto the strand. This always occurs when a DNA polymerase molecule hits a primer that is base paired to a longer piece of DNA.




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