BME100 s2015:Group17 12pmL4

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Owwnotebook icon.png BME 100 Spring 2015 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

Name: Hannah Austin
Name: Warner Kostes
Name: Ivanna Revel
Name: Alexandria Morales
Name: Your name

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

(http://www.promega.com/resources/protocols/product-information-sheets/g/gotaqcolorless-master-mix-m714-protocol/)

  • DNA/primer mix, 8 tubes, 50μL each: Each mix contains a different template DNA. All tubes hae 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 tip or samples will be cross-contamianted
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: share by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G17 + Positive control none
G17 - Negative control none
G17 1-1 Patient 1, replicate 1 48611
G17 1-2 Patient 1, replicate 2 48611
G17 1-3 Patient 1, replicate 3 48611
G17 2-1 Patient 2, replicate 1 56653
G17 2-2 Patient 2, replicate 2 56653
G17 2-3 Patient 2, replicate 3 56653


DNA Sample Set-up Procedure

  1. Step 1 : Label all the PCR mix test tubes
  2. Step 2 : Take the micropipettor and put on a fresh tip
  3. Step 3 : Carefully pipette the positive control into the tip
  4. Step 4 : Push the positive control into the PCR mix labeled "Positive Control"
  5. Step 5 : Repeat steps 1-4, with each DNA sample and the negative control


OpenPCR program

--Thermal Cycling 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

Q.1 What is the function of each component of a PCR reaction?

A PCR reaction is made up of several components, such as template DNA, primers, tag polymerase, and Deoyribonucleotides.Each of these components have a unique function. Template DNA is what the newly generated DNA is replicated from (serves as a blueprint for PCR). Primers attach to the specific sites on the DNA strangs that you want to copy. Tag polymerase carries out the replication process, like typical DNA polymerase, but Tag tolerates heat for the PCR. Finally, deoxyribonucleotides provide the PCR process with the "building blocks" to create new, replicated DNA.

Q.2 What happens to the components (listed above) during each step of thermal cycling?

There are six steps to thermal cycling. The initial step, where the temperature is 95 °C for 3 minutes, heats up the DNA preparing it for the imminent denaturation. Then, the denaturing step (where its 95°C for 30 seconds) is where the DNA strands are split from each other. Then, the annealing step, where the temperature is 57°C for 30 seconds, happens before the two strands of DNA template rejoin, and where the primers are attached to the target sites. The next step is to extend (72°C for 30 seconds). This is where the tag polymerase is activated, which then finds the primers. The final step (72°C for 3 minutes) is where the tag polymerase begins adding deoxyribonucleotides until it reaches the end of the strand. The final hold (at 4°C) is when the overal PCR reaction comes to a halt.

Q.3 DNA is made up of four types of molecules called nucleotides, designated as A, T, C, and G. Base-pairing, driven by hydrogen bonding, allows base pairs to stick together. Which base anneals to each base listed below?

  • Adenine (A) - Thymine (T)
  • Thymine (T) - Adenine (A)
  • Cytosine (C) - Guanine (G)
  • Guanine (G) - Cytosine (C)

Q.4 During which two steps of thermal cycling does base-pairing occur? Explain your answers.

Base-pairing occur between the steps of Annealing and Extending. During Annealing, the temperature is dropped to 57*C, which allows for the primer to attached to the separated DNA strands. The temperature is then raised to 72*C which enables the DNA polymerase to attach to the primer. Then, the DNA polymerase acts as the expediter to attached complementary nucleotide bases to the corresponding bases in the original DNA strand. Annealing and Extending combined act as the steps for base-pairing to occur.