BME100 f2014:Group12 L4

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Owwnotebook icon.png BME 100 Fall 2014 Home
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Jorge Espinoza
Devon Rusk
Darci Botsch
Peter Hillebrand
Krishnaa Pradhan
Sebastian Fonseca

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. ([1])
  • 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
G12 + Positive control none
G12 - Negative control none
G12 1-1 Patient 1, replicate 1 50958
G12 1-2 Patient 1, replicate 2 50958
G12 1-3 Patient 1, replicate 3 50958
G12 2-1 Patient 2, replicate 1 95193
G12 2-2 Patient 2, replicate 2 95193
G12 2-3 Patient 2, replicate 3 95193

Description of image

1. The qualitative data in this lab is the color of the samples. The quantitative data is the volume measurements of the samples in μl.

2. Accuracy reflects how close a measurement is to an accepted value while precision reflects how close measurements are to each other.

3. {need results}


DNA Sample Set-up Procedure

  1. Gather all materials needed for Lab A.
  2. In order to have two strips of four tubes, cut empty PCR tube strip in half. Then label each tube with proper tube labels.
  3. Once the tubes are placed in the rack, use the micropipette to transfer 50 μL of PCR reaction mix into the empty tube labeled as the positive control. It is important to make sure there is no cross-contamination of samples. Tips should not be re-used and should be discarded in the collection cup.
  4. The positive control DNA primer mix should now be transferred into the same tube. This will make the total volume in the positive control PCR reaction tube 100 μL.
  5. Repeat the two previous steps for the negative control, patient 1 replicates 1, 2, and 3, and patient 2 replicates 1, 2, and 3. The appropriate DNA primer mix must be used for its corresponding tubes.
  6. Now that all labeled tubes contain DNA primer mix and the PCR mix (100μL of complete PCR reaction), the lids should be closed tightly so they can be put unto the heating block.
  7. Once tubes are in the heating block slots (along with the other groups' tubes so that all slots are filled), the heating block can begin to run.

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

During a PCR reaction, the function of the template DNA is to provide the pattern for the sequence of nucleotides in an RNA transcript. Each gene has two DNA strands that make up that particular gene. The function of the primers during the reaction is to attach to specific sites on the DNA strands on either end of the segment that will be copied. Before the unraveled DNA strands can pair up, the primers attach to the specific part of the segment, in order to be able to replicate the DNA. During a PCR reaction, the Taq polymerase locates the primer which is attached to a single strand of DNA. After locating a primer, the Taq polymerase then begins to add complementary nucloetides on the strand until the end of the strand in order to complete the duplicated DNA strand. The deoxyribonucleotides (dNT's) are the single units of DNA. Each deoxyribonucleotide includes a nitrogenous base, a deoxyribose sugar,and one phosphate group. During the PCR reaction, deoxyribonucleotides will polymerize to form DNA. The phosphate group from one nucleotide will bond to the 3' carbon on another nucleotide, forming a bond within the DNA.


What Happens During Each Step of Thermal Cycling

During the initial step in the PCR reaction, the thermal cycler heats to 95 degrees Celsius for 3 minutes. During the 3 minutes the Template DNA, primers, Taq polymerase, and the deoxyribonucleotides (dNT's) all heat up in order for the DNA to be able to separate. In Denature, the temperature stays at 95 degrees Celsius and the double stranded DNA separates into two strands. After the DNA is separated into two single strands, the thermal cycler cools down to 57 degrees Celsius for 30 seconds. In this step, Anneal, the primers then attach to complementary matches on the target DNA sequence, in order to be ready to replicate. Then the thermal cycler heats up to 72 degree Celsius for 30 seconds in the Extend step. During this step, the Taq polymerase attaches to the primed sequences, and then adds nucleotides in order to extend the second stand of the DNA. This then completes the first cycle. These steps are repeated in subsequent cycles in order to create more DNA copies. After the 35 steps of the PCR reaction are completed, the mixture is held at a constant 72 degrees Celsius for 3 minutes during the final step in order to complete the extension process of the DNA. Finally, the mixture is held at 4 degrees Celsius during the final hold until it is removed from the thermal cycler and refrigerated for further use.


DNA Base Pairs

The base Adenine (A) pairs with the base Thymine (T). The base Cytosine (C) pairs with the base Guanine (G).


Thermal Cycling and Base Pairs

During the PCR reaction, base pairing occurs during both the anneal and extend steps. In the anneal step, there is a specific sequence of nucleotides that are found within the primer sequences that are connected to their corresponding sequences in the template DNA. The rules of base pairing, A pairs with T, and C pairs with G, allow for this to occur in the anneal step. In the extension step, the Taq polymerase is used to pair the deoxyribonucleotides to their corresponding base on the template DNA in order to complete the sequence. During both of these steps, base pairing occurs in order to complete the copied DNA sequence.