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OUR TEAM
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)
- 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
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| G7 1-3 |
Patient 1, replicate 3 |
89545
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| G7 2-1 |
Patient 2, replicate 1 |
25254
|
| G7 2-2 |
Patient 2, replicate 2 |
25254
|
| G7 2-3 |
Patient 2, replicate 3 |
25254
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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
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| 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:
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Separates the Template DNA into two separate strands
|
| Denature at 95°C for 30
seconds:
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Denatures the double stranded DNA into single strands
|
| Anneal at 57°C for 30
seconds:
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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
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| FINAL HOLD: 4°C: |
The DNA finishes it's replication process and stays constant
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Q3: Which base anneals to each base listed below?
| Adenine (A) |
Thymine (T) |
Cytosine (C) |
Guanine (G)
|
| T |
A |
G |
C
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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
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.
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BME 100 Fall 2016
