BME100 f2014:Group14 L4

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Contents

OUR TEAM

Fatima Sanchez Garcia
Fatima Sanchez Garcia
Claudia Czekaj
Claudia Czekaj
Ciaran McGirr
Ciaran McGirr
Taylor Brown
Taylor Brown
Lemlem Brook
Lemlem Brook
Brianna Steele
Brianna Steele

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: Mix contains 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.
  • 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 22157
G14 1-2 Patient 1, replicate 2 22157
G14 1-3 Patient 1, replicate 3 22157
G14 2-1 Patient 2, replicate 1 49384
G14 2-2 Patient 2, replicate 2 49384
G14 2-3 Patient 2, replicate 3 49384


DNA Sample Set-up Procedure#

  1. Retrieve the materials that are to be used in PCR: Positive sample, Negative sample, 3 samples from 2 patients, PCR mixture, 8 empty PCR Tubes
  2. Cut the row of empty PCR tubes in half, thus ensuring that there are two sets of 4 empty PCR tubes.
  3. Label the tubes according to labels listed in table. i.e: The positve sample would be labeled "G14p"
  4. Return the labeled tubes to the rack.
  5. Beginning with the positive sample, use proper pipetting technique to transfer 50μL of PCR reaction mix to the tube labeled "Positive". To prevent cross-contamination, discard tips after each transfer.
  6. Add 50μL of the positive sample to this same tube. There should now be a total volume of 100μL in the tube.
  7. Repeat the previous two step for the negative sample as well as all three of the patients' samples. Place the DNA samples with the corresponding tube. Every tube should have a volume of 100μL at the end of this process.
  8. Secure the lids on the prepared PCR reaction tubes.
  9. The prepared samples are now ready to be placed in PCR machine. Each group will occupy 8 slots, and two groups will share a PCR machine. Be sure to fill all 16 slots in PCR machine before beginning the reaction.


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

The Function of Each Component of a PCR Reaction:

The purpose of the Template DNA is to be the original DNA for replication. Without original DNA, there would be no way to select specific sections for replication, since all DNA is replicated from existing DNA. Small samples of Template DNA can be extracted from blood, skin or hair follicles. Primers are small synthesized sections of DNA that are designed to attach to a certain sequence of base pairs on the Template DNA. They single out the section of the Template DNA for replication and the enzyme Taq Polymerase uses it as a tag to start replication. Without the primers, Taq polymerase has nowhere to attach so no DNA replication would occur. Primers bond to specific sequences of he Template DNA based off of base pairs. Since A binds with T and C binds with G, the specific primer sequence will match up with the specific DNA pair, singling out the targeted gene. The purpose of the enzyme Taq polymerase is to replicate the specific part of the Template DNA. It attaches to the primers and begins to synthesize a replicated strand of DNA once the PCR is heated to 72 °C. Taq polymerase is found in bacteria that live in extreme heat conditions making it resistant to denaturing by the PCR heat cycles. It is not found in humans. When Taq polymerase is activated, it does not stop synthesizing the new strand until it reaches the end of the Template strand. Deoxyribonucleotides are the A T C and G nucleotides that make up the DNA. Since A binds with T and C binds with G, the Taq Polymerase uses the excess deoxyribonucleotides to match up to the Template DNA. Deoxyribonucleotides include all the sections of the DNA including the nitrogen base, the deoxyribose sugar and the nucleotide. These three components comprise the whole strand of DNA and are necessary to make the replicated strands.

Component Behavior During Steps of Thermal Cycling:

During the initial step where the reaction is heated to 95°C for 3 minutes to heat the PCR machine. Then, the Template DNA is initially denatured for 30 seconds meaning the two strands that make up the DNA separate. The primers, Taq Polymerase and the Deoxyribonucleotides are inactive at this stage. The next step is to Anneal at 57 °C for 30 seconds. This cooling step allows the primers to attach to the Template DNA and because there are so many primers in the solution, the primers attach more quickly to the Template than the two strands of DNA could recombine. The Taq Polymerase and Deoxyribonucleotides are still inactive at this stage. The next step is to Extend at 72 °C for 30 seconds which allows the Taq Polymerase to start replicating using the Primers as a starting point. The Taq Polymerase builds the replicated strand of DNA using the Deoxyribonucleotides in the solution. These steps are repeated 35 times. The Final Step is 72 °C for 3 minutes ensures that every Template strand has been replicated and the Final Hold preserves the structure of the DNA.

Base Pairs:

Adenine (A) binds with Thymine (T) and Cytosine (C) binds with Guanine (G.) During the Anneal and Extend steps of PCR, base pairing occurs. In the Annealing step, the primers pair up with the Template strands and during the Extending step, Taq Polymerase pairs the Deoxyribonucleotides with the Template strands to synthesize the new strand.





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