BME100 s2015:Group8 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: Priscilla Hernandez
Name: Alwaleed Alsahafi
Name: Victoria Sanford
Name: Sarah Soaf
Name: Taylor Simone Woods

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/gotaq- colorless-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


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G# + Positive control
G# - Negative control
G# 1-1 Patient 1, replicate 1 62525
G# 1-2 Patient 1, replicate 2 62525
G# 1-3 Patient 1, replicate 3 62525
G# 2-1 Patient 2, replicate 1 86787
G# 2-2 Patient 2, replicate 2 86787
G# 2-3 Patient 2, replicate 3 86787


DNA Sample Set-up Procedure

  1. Retrieve two patient samples (with given patient ID) and negative and positive control from the TA. The negative control is be a patient who has tested negative to having the SNP gene and the positive control is a patient who has tested positive. There will be three tubes for each patient.
  2. Make labels for the tubes with your group number and the corresponding label patient replicate. For example, this group's would label patient one's first replicate G82 1-1.
  3. Write down the patient ID's in their proper location in the table, but not on the tubes.

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

  PCR consists of five steps and a holding phase,  three of the steps are cycled 35 times. A PCR solution will require four main 

ingredients. The first ingredient that it will require is a template DNA source. The source nor the organism that it came from

matters but purer DNA yields higher results. Primers which are short single-strand segments of DNA are necessary to bind to the

complementary segment of the single-stranded DNA and for Taq polymerase to extend upon, thus replicating and amplifying the sought

after DNA segment. The final component deoxyribonucleotides or dNTP's are needed for the polymerase to base pair the template strand

with.Taq Polymerase which has been derived from a thermophile is used instead of regular DNA polymerase because it works optimally

at 72 degrees Celsius which is the optimal temperature for the extension step. Most PCR solutions will also contain a reaction

buffer in order to maintain optimal pH which will also maximize yields.


   During the first initial step, the PCR mix will be held at 95 degrees Celsius for 3 minutes. The high heat breaks the hydrogen 

bond that exist between the two stands of DNA leaving them as two single strands of DNA. This step is only repeated once. The next

three steps are each 30 seconds and are cycled 35 times, starting with the denature step at 95 degrees Celsius which seems

repetitive the first time but will serve to separate the primers from the single strand DNA so that the the next two steps may once

again occur. In the anneal step the mix is held at 57 degrees Celsius so that the primers may bond to their complementary DNA

segment. Two primers are made so that first, oriented in the forward direction and the second, in the reverse direction will contain

the segment meant for amplification in between. The final cycled step is the extension step which is at 72 degrees Celsius, in this

step Taq polymerase binds to the two primers and starts extending them. This extension is done by Taq polymerase grabbing a free

floating dNTP that complements the base immediately following the primer and bonding it with the template strand. This is repeated

until it meets the Taq polymerase coming in the opposite direction. The final extension step which is once again 72 degrees Celsius

but for three minutes lets any partially replicated strands finish. It is held at 4 degrees Celsius until retrieval time.


     Taq polymerase follows very strict restrictions for base pairing. In other words it doesn't match randomn nucleotides. Purines 

are always matched with a pyrimidine. In specific the purine adenine bonds with the pyrimidine thymine and the purine guanine base

pairs with the pyrimidine cytosine.