BME100 f2014:Group1 L4

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Contents

OUR TEAM

Name: Hawley Helmbrecht
Name: Hawley Helmbrecht
Name: Sarah FakhouryRole(s)
Name: Sarah Fakhoury
Role(s)
Name: Prerna GuptaRole(s)
Name: Prerna Gupta
Role(s)
Name: Jonathan Riecker
Name: Jonathan Riecker
Name: Michael PinedaRole(s)
Name: Michael Pineda
Role(s)
Name: Timothy Black
Name: Timothy Black

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat
  • Disposable Gloves
  • 8 tubes, 50μL each of PCR reaction mix: Contains a mixture of taq DNA polymerase, dNTP's and MgCl2
  • 8 tubes, 50μL each of DNA/primer mix: All tubes contain a different template of DNA, but the same forward and reverse primer
  • A strip of empty PCR tubes
  • Disposable pipette tips: only use each once. Never re-use disposable pipette tips or samples will be cross-contaminated
  • Cup for the discarded tips
  • Micropipettor
  • Open PCR machine


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G1 + Positive control none
G1 - Negative control none
G1 1-1 Patient 1, replicate 1 43236
G1 1-2 Patient 1, replicate 2 43236
G1 1-3 Patient 1, replicate 3 43236
G1 2-1 Patient 2, replicate 1 55773
G1 2-2 Patient 2, replicate 2 55773
G1 2-3 Patient 2, replicate 3 55773


DNA Sample Set-up Procedure

  1. Split the row of 8 PCR tubes in half
  2. Place the tubes in the rack
  3. Label the tubes according to DNA/primer mix (positive, negative, 1-1, 1-2, 1-3, 2-1, 2-2, 2-3)
  4. Set the micropippettor to 50μL
  5. Transfer 50μL of PCR reaction mix into each of the eight empty tubes
  6. Obtain a fresh pipette tip
  7. Transfer 50μL of positive control DNA/primer mix into the correctly labeled tube with PCR reaction mix in it
  8. Repeat steps 6 and 7 with the negative control, patient 1 replicates, and the patient 2 replicates. Making sure that they go into the correctly labeled tubes
  9. Take the tubes to the PCR machine
  10. Place tubes in the Thermal cycler


PCR Diagram (Bonus)

Polymerase Chain Reaction Process Resulting in the Amplification of a single DNA strand

Polymerase Chain Reaction Process Resulting in the Amplification of a single DNA strand

OpenPCR program

The thermal cycling program is used to amplify segments of DNA via the PCR. It heats and cools the DNA in cycles. It has two parameters including Primer Annealing as well as Primer Extension. Primer Annealing depends on the length, base composition, as well as concentration of the amplification primers. Extension depends upon the length and concentration of the target sequence and temperature. The optimum number of cycles depends mainly on the starting concentration of the target DNA. The optimum temperature for denaturation is about 90-95 degrees Celsius.

Description of image




Research and Development

PCR - The Underlying Technology

Template DNA within a PCR reaction is copied or amplified throughout the process in order to create billions of identical, specific DNA sequences that can be used to diagnose diseases, identify people based on DNA (i.e. criminals or relatives), or to identify different types of viruses or bacteria. In the PCR reaction as the DNA is duplicating, a primer, short piece of laboratory made DNA, binds to the template DNA in order to start the copying process. Taq Polymerase, a complex of proteins that occurs naturally and copies the DNA before the DNA divides in two, attaches to the end of the primers and starts to add nucleotides. The deoxyribonucleotides are four "building blocks" that make up DNA molecules. They come in four different types and A, C, G, and T. There purpose is to store and transmit the genetic information to form the duplicate DNA in the PCR reaction.

Initial Step for 3 minutes: 95°C Ensures all template DNA is denatured into two single stranded DNA molecules.

35 cycles of:

95°C 30 seconds of Denaturation: The templete DNA is split into two single stranded DNAmolecules.

57°C 30 seconds of Annealing: The forward and reverse primers bind to their appropriate nucleotide sequences on both single stranded DNA molecules.

72°C 30 seconds of Extension: Taq polymerase binds to the primer and begins to add corresponding nucleotides(dNTPs) to the single stranded DNA strand creating two double stranded DNA for each one DNA strand that we began with.

Final Step 72°C for 3 minutes: Continues extension to ensure that all single stranded DNA is fully extended.

Final Hold 4°C: To ensure that bonding occurs within the strands of DNA.

Pair molecules:

A(Adenine) binds to T(Thymine)

G(Guanine) binds to C(Cytosine)




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