LAB 2 WRITE-UP
SYBR Green Dye
SBYR green dye is a nucleic acid-sensitive gel dye that is useful in pinpointing double stranded DNA in samples within open PCR machines and other utilities such as the single-drop fluorimeter.
The single-drop fluorimeter is a fluorescence-based DNA detection device that aids in bringing to light PCR-Amplified DNA. The DNA sample is placed on a slide upon the machine and shined with a blue LED light. When the light hits the sample, the sample's DNA emits a dim green glow. From there, under a controlled, light-limited environment, a camera (specifically a camera on a phone) can be used to capture the green light.
How the Fluorescence Technique Works
The overall procedure of the fluorescence technique consisted of an extremely hydrophobic (water-hating) surface. This was used in order to not allow any water to be absorbed during the experiment. The slide atop was coated with an adhesive cover in order to keep the molecules in place during analysis, and keep them a certain distance from the slide at the same time. This being so, it alloweed for the blue LED light to be focused in the drop; causing an increase in intensity which helped create the fluorescent environment. In addition, The SBYR Green dye that was added with the solvent helped form a complex with DNA that preferred to stick to the surface than anywhere else on the Fluorimeter. If a water-soluble dye was used instead, the outcome of the experiment would differ because instead of the green glow, a colorful beam of light would have shown through.
- Note that the group used SYBR Green due to the fact it seemed to be the most eco-friendly."
SYBR Green 200mL
DNA Sample Solution
Micropipette and Tips
Smart Phone Camera Settings
- Iphone 5
- Flash: None
- ISO setting: NA
- White Balance: NA
- Exposure: NA
- Saturation: NA
- Contrast: NA
The iPhone was placed in a cradle in a way that made the lens the same height of the fluorimeter. In order to make them the same height, glass platforms were placed to raise the height of the fluorimeter. The distance was around 8 cm. The whole system was placed under the dark box and the pictures were taken. This was repeated for each new solution.
Photo of Fluorimeter
- Distance between the smart phone cradle and drop = 8cm
Solutions Used for Calibration
| Calf Thymus DNA solution concentration (microg/mL) || Volume of the 2X DNA solution (uL) || Volume of the SYBR GREEN I Dye Solution (uL) || Final DNA concentration in PicoGreen Assay (ng/mL)
| 0 || 80 || 80 || blank
| .25 || 80 || 80 || .125
| .5 || 80 || 80 || .25
| 1 || 80 || 80 || .5
| 2 || 80 || 80 || 1
| 5 || 80 || 80 || 2.5
Placing Samples onto the Fluorimeter
- Obtain waterproof slides, a Micropipette, and vials of various DNA samples along with a 200 ml vial of SYBR Green solution.
- Insert slide into fluorimeter
- Turn on blue fluorescent light and make sure that the beam is shined between the first two rows of the dotted slide.
- Take 80 micro-liters of SYBR Green solution into micropipette
- Release all of the 80 micro-liters of SYBR Green fluid onto the slide, then ensure that the beam of light hits the droplet.
- With a new plastic micropipette, take another 80 micro-liters of DNA Sample and extract into the SYBR Green droplet. Make sure the drops form a ball and doesn't spread around making individual droplets all over the slide.
- In order to see the glow of the SYBR Green with the DNA sample solution, a dark environment must be in place to see the dim green light. Place a box casing over the machine and take multiple pictures of the glowing solution.
- After the photo shoot is complete, and pictures are saved onto the phone, calibrate the micropipette to 160 micro-liters and extract all of the sample on the slide.
- Repeat the same steps for the additional DNA Samples.
- Clean Up Workstation
Representative Images of Samples
- Sample before DNA was present
- Sample After DNA was present
Image J Values for All Samples [See worksheet page 5]
| DNA Final Concentration (ng/mL)|| Average Mean|| Average IntDens
| 5 || 106.75 || 4083499
| 2 || 95.35 || 4017729
| 1 || 79.58 || 3390288
| .5|| 54.68 || 2879054
| .25 || 45.90 || 1859347
| 0 || 33.69 || 994680
Fitting a Straight Line