BME100 s2015:Group14 12pmL4

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OUR TEAM

Name: Nathan Francois
Name: Lexi Bounds
Name: Dakota Graham
Name: Thor Kissman
Name: Justin Mieth

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
G14 + Positive control none
G14 - Negative control none
G14 1-1 Patient 1, replicate 1 80565
G14 1-2 Patient 1, replicate 2 80565
G14 1-3 Patient 1, replicate 3 80565
G14 2-1 Patient 2, replicate 1 87710
G14 2-2 Patient 2, replicate 2 87710
G14 2-3 Patient 2, replicate 3 87710


DNA Sample Set-up Procedure

  1. Put on safety equipment.
  2. Label 8 empty PCR tubes according to the table above.
  3. Using a micropipetter, take a clean, disposable pipette tip and draw 50 uL of PCR reaction mix into an empty PCR tube. Discard the pipette tip.
  4. With a new pipette tip, draw 50 uL of DNA/primer mix into the PCR tube. Dispose of the pipette tip.
  5. Repeat the previous steps with the 7 remaining DNA/primer mixes and the 7 remaining PCR tubes.
  6. Place the PCR tubes into the PCR machine and close the lid.
  7. Set up the PCR program you plan to run.
  8. Make sure that the lid is heated to 100 degrees Celsius before the initial step.
  9. For the initial step of the PCR, change the temperature to 95 degrees and allow the tubes to heat for two minutes.
  10. Begin PCR cycles. For each cycle, denature the DNA at 95 degrees Celsius for 30 seconds, cool it at 57 degrees Celsius for 30 seconds, and finally allow DNA to replicate at 72 degrees Celsius for 30 seconds.
  11. Complete 35 PCR cycles.
  12. When 35 cycles are completed, set the temperature at 72 degrees Celsius for 2 minutes.
  13. Last, set the temperature to 4 degrees Celsius before removing the PCR tubes.

OpenPCR program

Open PCR Program Main Screen
PCR steps used in this experiment
PCR steps used in this experiment, cont'd.



Research and Development

PCR - The Underlying Technology

A PCR takes a target sequence of DNA and exponentially increases the quantity of that target sequence. There are four main molecules needed for a PCR:

  • Template DNA
  • RNA primers
  • Taq DNA polymerase
  • Deoxyribonucleotides (monomer building blocks of DNA)

In a PCR, the template DNA is the DNA strand that has the target DNA sequence in it. The RNA primers in a PCR are short RNA strands that have a specific sequence. There are two primers needed to get a target DNA sequence to replicate in full. RNA primers have a specific ribonucleotide sequence that allows them to bind to a template DNA at a desired target sequence. Genetic technology allows specific RNA primer strands to be synthesized which can base pair with targeted DNA sequences. This makes sure that only target DNA can be replicated. Primers are needed to allow for DNA replication. Taq DNA polymerase cannot bind to a DNA molecule and start replication if there is not a primer for it to bind to. Once bound, Taq polymerase elongates the DNA sequence moving from the 5' end to the 3' end. Taq polymerase cannot replicate, however, without the presence of deoxyribonucleotides. These are the individual building blocks of DNA molecules. There are 4 types of deoxyribonucleotides found in DNA. These are Adanine (A), Guanine (G), Cytosine (C), and Thymine (T). In DNA, A always binds with T, while C always binds with G. These are called base-pairs. When Taq polymerase replicates DNA, it matches up the nucleotides it is adding to the corresponding base-pair.

PCRs run in a cycle. In the initial step, the PCR tubes are heated at 95 degrees Celsius for 2-3 minutes. In this step, the DNA inside the test tubes begins to denature, which allows for primers to bind to the DNA and for polymerase to begin replication. The cycles then begin. During the Denature step, the PCR tubes are heated at 95 degrees Celsius for 30 seconds. Just like the initial step, this step makes sure that all DNA strands are cleaved in half so that replication can occur. During the Anneal step, the PCR tubes are cooled to 57 degrees Celsius for 30 seconds. In this step, RNA primers bind to target DNA sequences through base-pairing. The 30 seconds is a control to make sure that all of DNA has been bound by primers. For the Elongation step, the PCR tubes are heated to 72 degrees Celsius for 30 seconds. During this step, Taq polymerase binds to the DNA at the site of the primers. Deoxyribonucleotides are then bound together into a new DNA strand which is made up of the opposite bases from the template strand. Base-pairing in this step results in the replicated DNA strands. The cycle then starts over at the Denature step. After the desired number of cycles are completed, the PCR tubes are kept at 72 degrees Celsius for 2-3 minutes. This step ensures that all of the DNA has been replicated and that the process has completed. Finally, the tubes are cooled to 4 degrees Celsius. Doing so prevents the chance that the PCR reaction will continue longer than desired.

When everything has been completed, the amount of DNA in the test tubes can be millions to billions of times larger than the amount initially present.