BME100 f2016:Group7 W1030AM L4

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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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Contents

OUR TEAM

Name: Koop Bills
Name: Koop Bills
Name: Tyler Lent
Name: Tyler Lent
Name: Israel Zaldivar
Name: Israel Zaldivar
Name: Omar Maranon
Name: Omar Maranon
Name: Maria Hanna
Name: Maria Hanna

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coats
  • Disposable Gloves
  • PCR reaction mix:
8 50 μL tubes (each must contain Taq DNA polymerase, MgCL_2, and dNTP's)
  • DNA/ primer mix
8 50 μL tubes (each must contain a different template DNA with the same forward & reverse primer)
  • One strip of empty PCR tubes
  • Disposable pipet tips
  • Cup for discarded tips
  • Micropipettor
  • OpenPCRmachine


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G7 + Positive control none
G7 - Negative control none
G7 1-1 Patient 1, replicate 1 89545
G7 1-2 Patient 1, replicate 2 89545
G7 1-3 Patient 1, replicate 3 89545
G7 2-1 Patient 2, replicate 1 25254
G7 2-2 Patient 2, replicate 2 25254
G7 2-3 Patient 2, replicate 3 25254


DNA Sample Set-up Procedure

It is imperative that whenever you micropipette anything that you dispose of the tube into the labeled empty cup for used tips, otherwise you may cross contaminate the sample
  • Put on PPE
  • Label empty PCR strip according to the above table
  • Into the tube labeled G7 P micropipette the given positive control
  • Into the tube labeled G7 N micropipette the given negative control
  • Into the tube labeled G7 1-1, 1-2, and 1-3 micropipette some of the sample from patient 1
  • Into the tube labeled G7 2-1, 2-2, and 2-3 micropipette some of the sample from patient 2
  • At this point you will begin to add in appropriate DNA primer mixes based off of the labels
(postive, negative, patient 1, and patient 2)
  • Next you will begin to add in appropriate PCR reaction mix into the tubes
  • At this point you may close all lids to the PCR tubes and insert them into the thermal cycler!!

OpenPCR program
HEATED LID: 100°C
INITIAL STEP: 25

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 PCR reaction?

Template DNA: The Template DNA provides the piece that is used in the replication of a certain segment
Primers: Primers are used to determine where the Taq Polymerase should begin
Taq Polymerase: The Taq Polymerase uses the process of transcription in order to bind free floating nucleotides to the single strand of DNA
Dioxyribonucleotides (dNTP's): Diooxyribonucleotides are the available nucleotides in a solution so that they may be bonded to the single stranded DNA

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

INITIAL STEP: 95°C for 3

minutes:

Separates the Template DNA into two separate strands
Denature​ at 95°C for 30

seconds:

Denatures the double stranded DNA into single strands
Anneal​ at 57°C for 30

seconds:

The primers attach to their designated spots on each of the single strands from the template DNA
Extend​ at 72°C for 30

seconds:

The Taq Polymerase binds to the primers and connects the nucleotides together in order to complete the single stranded template DNA
FINAL STEP: 72°C for 3

minutes:

Similar to the previous step, but it allots more time for the Taq Polymerase to work
FINAL HOLD: 4°C: The DNA finishes it's replication process and stays constant

Q3: Which base anneals to each base listed below?

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

Q4: During which two steps of thermal cycling does base-pairing occur?
Answer: Base-pairing occurs when the primers bond to the single strand of DNA and when the Polymerase binds the nucleotides to the strand of DNA

SNP Information & Primer Design

Background: About the Disease SNP

SNP or single nucleotide polymorphism is caused by the variation of a single nucleotide ( an A, T, G, or C) in a specific loci in the genome. Another quality of SNP is that the replacement must occur at a specific base pair in a small population and thus differ from the accepted norm of the general population. These small variations affect several aspects of the "human condition" especially susceptibility to disease. Another interesting note is that SNP's may affect the cell by causing an overproduction of a certain protein important to the gene that has the variation and thus cause complications (though this isn't always the case).


Primer Design and Testing


The results of our test indicate that we were able to correctly identify primers and that the primers that we came up with did in fact link to the correct gene sequence in the database. We were also able to correctly produce a mutated sequence that did not appear in the database because that database was reflective of what the gene seuence should correctly be. This was an important discovery because we can now be sure that the database has only valid results. Image:Correct.jpg

Image:Wrong.jpg

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