BME100 s2014:W Group11 L5
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LAB 5 WRITE-UP
SYBR Green Dye
SYBR Green I is a fluorescent dye used for binding double-stranded DNA so that quantification and visualization is possible. SYBR Green is a synthetic, asymmetrical cyanine dye that was designed to work throughout a wide range of temperatures, bind in a way that does not interfere with enzymes such as DNA polymerases and nucleases, and accurately quantify DNA in varying concentrations. Real time PCR is the most common use for the dye, where the fluorescence increases as each PCR cycle amplifies the DNA so you can visually observe amplification.
The Single-Drop Fluorimeter is a device designed to help detect any presence of double stranded DNA in a liquid sample by measuring its fluorescence. It is essentially a small plastic device with a space on top for a multi-welled slide. The slide is illuminated by a single blue LED light which is turned on by a switch located on the right hand side of the Fluorimeter. The slide of the fluorometer is positioned so that the blue LED can iluminate any drop placed over a designated well.
The fluorescence technique works by adding a fluorescent die to a liquid with an unknown percentage of double stranded DNA. The slides used in this analysis have a hydrophobic Teflon film on one side of the slide, designed to hold each sample in place. The film contains breaks which act as wells where liquids can be held in precise quantities. These breaks also keep each of the drops apart from one another so that the samples do not mix. All these traits make it easier to observe and analyze our samples when using the LED. When the samples are placed in the LED's range, any DNA stands at the top of the drop sized sample can be seen because the light causes them to fluoresce.
Smart Phone Camera Settings
Representative Images of Samples
Sample with DNA (Positive Control)
Sample with no DNA (Negative Control)
Image J Values for All Samples
Your positive control PCR result was 4.35 μg/mL
Your negative control PCR result was -0.146 μg/mL
Patient 10072: When DNA from this patient was combined with the SYBR green dye and viewed in the fluorimeter, it produced an average Intdens reading of 3.91 μg/mL
Patient 83576 : When DNA from this patient was combined with the SYBR green dye and viewed in the fluorimeter, it produced an average Intdens reading of -0.364 μg/mL
Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion.
Patient 10072 : Each of the samples from this patient very closely resembled the positive control. All drops glowed bright fluorescent green in the fluorimeter. This patient appears to have tested positively for the gene of interest because the SYBR green dye was able to attach to double-stranded DNA molecules and fluoresce. Since the dye was able to do this, the PCR reactions must have produced many copies of the target strand of DNA, meaning it was present in the samples from this patient.
Patient 83576 : Each of the samples from this patient very closely resembled the negative control. None of the drops glowed or fluoresced when viewed in the fluorimeter. This patient appears to have tested negatively for the gene of interest because the SYBR green dye did not bind to any double-stranded DNA molecules to fluoresce. Since no double-stranded DNA molecules were produced in the PCR reaction, the patient did not have the target sequence of DNA.