BME100 f2017:Group14 W1030 L4

From OpenWetWare
Owwnotebook icon.png BME 100 Fall 2017 Home
People
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
Course Logistics For Instructors
Photos
Wiki Editing Help
BME494 Asu logo.png


OUR TEAM

Name: Lydia Gabric
Name: Sarah Bacon
Name: Anshul Krishnan
Name: Charmayne Seaton
Name: Chris Ward

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
  • 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 reuse disposable pipette tips. If you do, the samples will become cross-contaminated.
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G14 + Positive control none
G14 - Negative control none
G14 1-1 Patient 1, replicate 1 28550
G14 1-2 Patient 1, replicate 2 28550
G14 1-3 Patient 1, replicate 3 28550
G14 2-1 Patient 2, replicate 1 55154
G14 2-2 Patient 2, replicate 2 55154
G14 2-3 Patient 2, replicate 3 55154


DNA Sample Set-up Procedure

  1. Step 1: Move DNA and Primer mix into a PCR tube
  2. Step 2: Add PCR reaction mix into the PCR tube with the DNA and Primer mix
  3. Step 3: Place PCR tube into a DNA Thermal Cycler
  4. Step 4: Follow the OpenPCR Program steps below and start Thermal Cycler

OpenPCR program

HEATED LID: 100°C
INITIAL STEP: 95°C for 2 minutes
NUMBER OF CYCLES: 25
Denature at 95° 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

The purpose of the Polymerase Chain Reaction is to amplify a targeted DNA Sequence by making several copies of it. The Polymerase Chain Reaction principally consists of primer mediated enzymatic amplification of the Deoxyribonucleic Acid. It is based on the ability of DNA polymerase to synthesize complementary DNA strands when exposed to a template strand.

The PCR reaction requires the following components in order for it to be carried out:

1. DNA Template: The desired DNA sequence to be amplified

2. DNA Polymerase: The enzyme that facilitates the reaction

3. Oligonucleotide Primers: Short pieces of complementary DNA strands

4. Deoxynucleotide Phosphates: to provide energy for the reaction and are also the building blocks of DNA synthesis

5. Buffer System: To provide optimal condition for the reaction to take place

There are 3 stages to the Polymerase Chain Reaction:

  • Denaturation: The first step of the PCR reaction is to heat the DNA strands to 94 degrees Celcius to denature the double-stranded DNA into single-stranded DNA, hydrogen bonds are broken between the strands
  • Annealing: The second step of the PCR reaction is to reduce the temperature to 54 degrees Celcius to let Primers to anneal to their complementary sequence in the template DNA
  • Elongation: The third step of the PCR reaction occurs at 72 degrees Celcius where the polymerase enzyme sequentially adds bases to the 3′ each primer, extending the DNA sequence in the 5′ to 3′ direction. Thus the new, duplicate strand is elongated


Citation: [1] “Polymerase Chain Reaction (PCR) : Principle, Procedure, Components, Types and Applications.” LaboratoryInfo.com, Laboratory Info, 28 July 2015, laboratoryinfo.com/polymerase-chain-reaction-pcr/.

SNP Information & Primer Design

Background: About the Disease SNP
The disease and SNP is found in Homo Sapiens on chromosome 19:44907853. The clinical significance of this SNP is pathogenic and it is linked Alzheimer’s. APOE stands for apolipoprotein E. It acts as a biomarker in the genes of Alzheimer’s disease and Subarachnoid hemorrhages. Alzheimer’s is often caused by the expression of an alternate gene, the allele. The disease-associated allele, in this case, contains the “T” codon with the numerical position of the SNP being 44907853.



Primer Design and Testing


In short, when we wrote our primer codes and ran them through the database, it revealed that the codes we wrote were located on chromosome 19. This is correct because it was revealed earlier that our gene was located on that very chromosome. Additionally, we ran our disease forward primer through the database, it revealed no results, which was the desired result.

BME100WG14 PCRlabpic1.PNG

BME100WG14 PCRlabpic2.PNG


Sources

“Polymerase Chain Reaction (PCR) : Principle, Procedure, Components, Types and Applications.” LaboratoryInfo.com, Laboratory Info, 28 July 2015, laboratoryinfo.com/polymerase-chain-reaction-pcr/.