BME100 f2014:Group21 L4

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Contents

OUR TEAM

Name: Dustin D. Burley
Name: Dustin D. Burley
Name: Mackenzie Konves
Name: Mackenzie Konves
Name: Scott Boege
Name: Scott Boege
Name: Nicholas Goettl
Name: Nicholas Goettl
Name: Travis Tibbs
Name: Travis Tibbs
Name: Lionel Davis
Name: Lionel Davis

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat
  • Disposable gloves
  • PCR reaction mix, 8 tubes, 50μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s
  • A strip of empty PCR tubes
  • Disposable pipette tipe
  • Cup for discarded tips
  • DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA.
  • OpenPCR machine
  • Micropipettor


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G21 + Positive control none
G21 - Negative control none
G21 1-1 Patient 1, replicate 1 17567
G21 1-2 Patient 1, replicate 2 17567
G21 1-3 Patient 1, replicate 3 17567
G21 2-1 Patient 2, replicate 1 57701
G21 2-2 Patient 2, replicate 2 57701
G21 2-3 Patient 2, replicate 3 57701


DNA Sample Set-up Procedure

  1. Gather all the materials listed above to start the experiment.
  2. Create two sets of four PCR tubes by splitting the 8 tubes into two columns.
  3. Label the different tubes according to the DNA template that will be placed in them. For example the third replicate of the first patient’s DNA would be labeled G21 1-3. There will be two controls (positive and negative), three copies for patient 1, and three copies for patient 2.
  4. Using the correct pipetting techniques that were practiced with the food coloring mixes, add 50µL of the PCR mix (which includes the Taq polymerase, primers one and two, and deoxyribonucleotides) to each tube.
  5. Add 50µL of the DNA sample to the tube with the correct label (example: Patient 1, replicate 1 should go in the tube labeled 1-1). BE SURE TO DISCARD PIPETTE TIPS AFTER EACH TRANSFER.
  6. Repeat step five for each of the DNA samples given and the two controls.
  7. Confirm that there are 100µL in each tube; 50µL of the DNA sample and 50µL of the PCR mix.
  8. Make sure the tubes are securely shut, and then added to the thermal cycler. Since two groups are sharing a machine, make sure that each of the 16 slots are occupied before running the machine.


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

  • Components of Polymerase Chain Reaction

The four main components of a PCR are Template DNA, Taq Polymerase, Primers, and Deoxyribonucleotides. Template DNA is the double stranded DNA that is to be amplified. It is a segment of someone or some organisms genome that wants be further studied, or it could be a strand of DNA that codes for a useful protein, like insulin. Taq Polymerase is a protein that is found in an organism, thermus aquaticus that lives in incredibly hot environments like hot springs. This enzyme is essential to PCR because of the high temperature that the reaction needs to occur at. The markers that show Taq Polymerase where to start and stop the DNA replication, and are what allow such specific strands to be targeted in PCR. Deoxyribonucleotides are Adenine, Thymine, Cytosine and Guanine. These are what make up the DNA and are what are paired together by Taq Polymerase. These are the parts of DNA that code for certain amino acids, which are the building blocks of proteins. The deoxyribonucleotides Adenine and Thymine pair together and Cytosine and Guanine pair together as well.


  • What Happens to these Components During the Reaction?

During PCR the four components listed above go through 6 different processes. The first step is to heat up the solution to 95°C for three minutes. This makes the solution hot to improve precision, because primers will not be able to bond to the template DNA in the wrong spots as it slowly heats up and splits in two. After the three minutes, the solution is kept at 95°C so the template DNA is denatured, and splits into two separate single strands of DNA. Once the DNA is denatured, it is cooled down to 57°C so that primers can bond/anneal to the two strands of DNA in the target region. This is the first time base-pairing occurs in the PCR. Primers have certain deoxyribonucleotides on them, so that they mirror the section of the DNA that is right before the strand that wished to be amplified starts and right after this region stops. These primers show the Taq Polymerase where to start and stop replication, and once they are bonded to the two strands of DNA the solution is heated backed up to 72°C. This is because Taq Polymerase works more efficiently at higher temperatures. During this phase, called “extension,” the Taq Polymerase quickly bonds the complementary base pairs (Adenine to Thymine and Cytosine to Guanine) to the strand of DNA. When this step is complete the replication of the original template DNA is complete, and there are two copies of it. This is the second time that base-pairing occurs during PCR. After this the solution is kept at 72°C for the final step to make sure that all of the targeted areas have been extended/replicated. After this the solution is kept at 4°C as a short term storage solution. The temperature is low so that there is little possibility of denaturation.


Extra Credit: Images to Help Understand PCR

picture 1

This picture was taken from http://bit.ly/1tJPyxx. This picture shows how and why primers are useful during a PCR.



picture 2

This picture was taken from http://bit.ly/1nOY3FB. This picture shows how Taq Polymerase is used to combine free deoxyribonucleotides to their complementary base-pairs on single strands of DNA





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