LAB 5 WRITE-UP
Smart Phone Camera Settings
- Type of Smartphone: iPhone
- Flash: Inactivated
- ISO setting: N/A
- White Balance: Auto
- Exposure: N/A
- Saturation: N/A
- Contrast: N/A
Settings that are N/A:
Camera is sensitive enough to get a reasonable calibration with DNA concentration
Camera was focused when necessary
- Turn on the camera on the smartphone and calibrate according to the settings above
- Place the smartphone in the cradle
- Place the cradle at a right angle to the slide
- Adjust the height of the fluorimeter using the plastic trays in order to take a picture of the drop sideways
- Adjust the distance between the cradle with the smartphone and the slide so that the cradle is at least 4 cm away from the slide
- Record the distance between the cradle and the slide with a ruler
- Distance between the smart phone cradle and drop = 8cm
Solutions Used for Calibration
- Water at pH = 8
- SYBR green I dye solution
- DNA solution
Placing Samples onto the Fluorimeter
- Use the pipettor to place a drop of the SYBR green I dye solution (80 microliters) in the middle of the first two rows of the slide
- Use the pipettor to add a drop of the water blank solution (80 microliters)
- Adjust the slide so that the drop focuses the blue LED light to the middle of the black fiber optic fitting on the other side of the drop
- Cover the fluorimeter and camera with the light box so that all stray light is removed
- Take three pictures and make sure the drop is focused
- Remove the box
- Use the pipettor to remove the drop from the slide (160 microliters)
- Move the slide to the next position
- Repeat steps 1-8 for the patients and the DNA solution
Representative Images of Negative and Positive Samples
Image J Values for All Calibrator Samples
PCR Results Summary
- Our positive control PCR result was 0.05596862783 μg/mL
- Our negative control PCR result was 0.02701367497 μg/mL
- Patient 11316 : There is a notable amount of the SYBR green I dye solution in the drop.
- Patient 25353 : Compared to patient 11316, the amount of SYBR green I dye solution in the drop is insignificant. There is still a significant amount present, however, patient 11316 shows more of the dye.
- Patient 11316 : It was concluded that the patient did not contract the disease because the PCR concentration is close to the the negative control PCR result.
- Patient 25353 : It was concluded that the patient did not contract the disease because the amount of dye present in the drop is less than the amount of dye present in the drop for patient 11316. Both the PCR concentrations in the drops are extremely close to the negative control value amount, however patient 25353 shows a lesser amount (closer to the negative control)
SNP Information & Primer Design
Background: About the Disease SNP
SNPs, or single nucleotide polymorphisms, are an affliction on one's DNA. They are the most common source of genetic variation found among HomoSapiens, or the human population.
Essentially a SNP takes one nucleotide and changes it into a different one. For example, if the nucleotide is supposed to be an A, an SNP will change the A into a T.
By changing nucleotides, variations within humans arise, or the differences in humans' phenotypes (appearances).
Question answers from the lab manual:
- Nucleotide- “A nucleotide is one of the structural components, or building blocks, of DNA and RNA.
A nucleotide consists of a base (one of four chemicals: adenine, thymine, guanine, and cytosine) plus a molecule of sugar and one of phosphoric acid.”
Taken from: http://www.ncbi.nlm.nih.gov/Class/MLACourse/Original8Hour/Genetics/nucleotide.html
- Polymorphism-“Single nucleotide polymorphisms, frequently called SNPs (pronounced “snips”), are the most common type of genetic variation among people.
Each SNP represents a difference in a single DNA building block, called a nucleotide.”
Taken from: http://ghr.nlm.nih.gov/handbook/genomicresearch/snp
- What species is the variation found in? – Homo Sapiens
- What chromosome is the variation located on? - 8:19956018
- What is listed as the Clinical significance of this SNP? – Pathogenic
- Which gene(s) is this SNP associated with? – LPL
- Click the PubMed link to view summaries of research associated with the SNP. What disease is linked to this SNP? – Metabolic syndrome susceptibility
- What does LPL stand for? – Lipoprotein lipase
- What is the function of LPL? To find out, click the LPL link. Look for “Gene ontology” in the right hand list and click it. Write the first three unique terms you see...
1) apolipoprotein binding
2) heparin binding
3) chylomic ron remodeling
- What is an allele? – “An allele is an alternative form of a gene(one member of a pair) that is located at a specific position on a specific chromosome. These DNA codings determine distinct traits that can be passed on from parents to offspring throughsexual reproduction.” Taken from: http://biology.about.com/od/geneticsglossary/g/alleles.htm
- The disease-associated allele contains what sequence? – A G T
- The Numerical position of the SNP is -19956018
- Non-disease forward primer (20 nt): A A T C T G G G C T A T G A G A T C A A
- The numerical position exactly 200 bases to the right of the disease SNP is: 19956219
- Non-disease reverse primer (20 nt): A A T G C A A C C C C C T A T C A A C A G
- Disease forward primer (20 nt): A A T C T G G G C T A T G A G A T C A G
- Disease reverse primer: same as non-disease reverse primer
Primer Design and Testing