BME100 s2015:Group6 9amL4

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OUR TEAM

Name: Sara Belko
Name: Luc Tieu
Name: Priscilla Delgado
Name: Al Imam
Name: Francisco Campa

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, 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

Patient IDs:

  • 1. 73039
  • 2. 40429

PCR Reaction Sample List

Tube Level PCR Reaction Patient ID
GP positivecontrol none
GN negativecontrol none
G1-1 Patient1Replicate1 73039
G1-2 Patient1Replicate2 73039
G1-3 Patient1Replicate3 73039
G2-1 Patient2Replicate1 40429
G2-2 Patient2Replicate2 40429
G2-3 Patient2Replicate3 40429

DNA Sample Set-up Procedure

  • 1. Collect materials
  • 2. Cut strip of 8 empty tubes into two sets of four to allow for easy placement into the PCR machine.
  • 3. Label each of the 8 tubes with a black marker: GP, GN, G1-1, G1-2, G1-3, G2-1, G2-2, G2-3.
  • 4. Place tubes back into the rack
  • 5. Pipet 50 micro-liters of PCR reaction mix into empty tube labeled GP (positive control). Throw away disposable tip.
  • 6. Using a new pipet tip, pipet 50 micro-liters of positive control DNA/primer mix into the same tube.
  • 7. Repeat steps 5 and 6 for the rest of the tubes using the appropriate DNA/primer mix.
  • 8. Close lids tightly
  • 9. Place tubes into the slots of the PCR machine where DNA will be replicated. Make sure your group can identify your samples from the other group's.

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

Q1. What is the function of each component of a PCR reaction?

Template DNA: sequence of DNA that contains the area interested in. This sequence undergoes extreme heat to denature so replication can take place.

Primers: single stranded DNA that is complementary to template DNA. They are synthesized from 5' to 3' to complement the template DNA (3' to 5').

Taq Polymerase: a heat-resistant enzyme that links to the fork in the template DNA and creates the primers to complement the template DNA.

Deoxyribonucleotides (dNTP's): base pairs of DNA (A,T,C,G). In RNA, U is a dNTP in place of T.

Q2. What happens to the components (listed above) during each step of thermal cycling?

Initial step: 95 C for 3 minute:

  • Denature at 95 C for 30 seconds:
  • Anneal at 57 C for 30 seconds:
  • Extend at 72 C for 30 seconds:

Final step: 72 C for 3 minutes:

Final hold: 4 C:

Q3. DNA is made up of four types of molecules called nuclotides, 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

Thymine (T): Adenine

Cytosine (C): Guanine

Guanine (G): Cytosine

Name: PCR Diagram

Picture References:

"DNA Replication." Wikipedia. Wikimedia Foundation. Web. 31 Mar. 2015. <http://en.wikipedia.org/wiki/DNA_replication>.