BME100 s2014:T Group8 L5: Difference between revisions
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'''How the Fluorescence Technique Works'''<br> | '''How the Fluorescence Technique Works'''<br> | ||
'' | ''Since using the naked eye to view if a sample has DNA is near to impossible; a fluorescent dye is used. | ||
A camera makes the florescent dye even easier to see, and creates images which can be analyzed for amount of dye present. | |||
The way the fluorescence works is by utilizing a green dye that activates only under the presence of double stranded DNA (or dsDNA). | |||
'' | |||
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Placing Samples onto the Fluorimeter: | |||
1. Turn on the flourimeter. Place the super hydrophobic slide on the flourimeter. | |||
On the hydrophobic side of the flourimeter, place a 80 microliter drop of SYBR GREEN I on the middle rows of the slide. | |||
2. Now on the sphere of dye that is on the slide, add 80 microliter of one of the solutions being tested. This will now react or not. | |||
3. Next the flourimeter will shine a blue light, align the slide so that the drop sample is in the middle of this light. | |||
4. Acquire a box that can cover the flourimeter in order to create a dark environment. | |||
Place a smartphone in the cradle, and use a timer on the camera to take a picture of the drop. Once the timer starts quickly put the box over the whole setup, so that outside light does not interfere. | |||
<br> | <br> | ||
Line 132: | Line 145: | ||
Low DNA - using green channel | Low DNA - using green channel | ||
[[Image:drop2.png]] | [[Image:drop2.png]] | ||
<br> | |||
{| cellpadding="2" style="border: 1px solid darkgray;" | |||
! width="140" | High DNA Concentration | |||
! width="150" | Low DNA COncentration | |||
|- border="0" | |||
| [[Image:Positie_controlgroup8dna.jpg|500px]] | |||
| [[Image:Negative_controlgroup8dna.jpg|500px]] | |||
|- align="center" | |||
| Positive Control || Negative Control | |||
|} | |||
<br> | |||
'''Image J Values for All Samples''' | '''Image J Values for All Samples''' | ||
Line 145: | Line 169: | ||
| align="center" style="background:#f0f0f0;"|'''RAWINTEDEN (drop) - RAWINTDEN (background)''' | | align="center" style="background:#f0f0f0;"|'''RAWINTEDEN (drop) - RAWINTDEN (background)''' | ||
|- | |- | ||
| rowspan= "6"| Trial 1 | | rowspan="6"| Trial 1 | ||
| ||0|| | | 0 | ||
| 91382 | |||
| 71.613 | |||
| 6544125 | |||
| 18757 | |||
| 6525368 | |||
|- | |||
| 0.25 | |||
| 83776 | |||
| 40.875 | |||
| 3424354 | |||
| 2865 | |||
| 3421489 | |||
|- | |||
| 0.5 | |||
| 64632 | |||
| 64.815 | |||
| 4189144 | |||
| 373 | |||
| 4188771 | |||
|- | |||
| 1 | |||
| 72068 | |||
| 74.307 | |||
| 5355152 | |||
| 2461 | |||
| 5352691 | |||
|- | |||
| 2 | |||
| 74956 | |||
| 116.601 | |||
| 8739909 | |||
| 8280 | |||
| 8731629 | |||
|- | |||
| 5 | |||
| 81146 | |||
| 164.265 | |||
| 13329423 | |||
| 5734 | |||
| 13323689 | |||
|- | |||
| rowspan="6"| Trial 2 | |||
| 0||84384 | |||
| 53.044 | |||
| 4476036 | |||
| 2665 | |||
| 4473371 | |||
|- | |||
| 0.25 | |||
| 60532 | |||
| 57.136 | |||
| 3458560 | |||
| 1701 | |||
| 3456859 | |||
|- | |||
| 0.5 | |||
| 88468 | |||
| 42.136 | |||
| 3727677 | |||
| 209 | |||
| 3727468 | |||
|- | |||
| 1 | |||
| 84626 | |||
| 85.175 | |||
| 7208044 | |||
| 106 | |||
| 7207938 | |||
|- | |- | ||
| || | | 2 | ||
| 87906 | |||
| 99.08 | |||
| 8709740 | |||
| 174 | |||
| 8709566 | |||
|- | |- | ||
| | | 5 | ||
| 89884 | |||
| 162.199 | |||
| 14579066 | |||
| 87 | |||
| 14578979 | |||
|- | |- | ||
| || | | rowspan="6"| Trial 3 | ||
| 0 | |||
| 97260 | |||
| 45.051 | |||
| 4381686 | |||
| 7168 | |||
| 4374518 | |||
|- | |- | ||
| || | | 0.25 | ||
| 92784 | |||
| 54.433 | |||
| 5050546 | |||
| 593 | |||
| 5049953 | |||
|- | |- | ||
| | | 0.5 | ||
| 88652 | |||
| 69.382 | |||
| 6150810 | |||
| 176 | |||
| 6150634 | |||
|- | |- | ||
| | | 1 | ||
| 111748 | |||
| 73.74 | |||
| 8240277 | |||
| 783 | |||
| 8239494 | |||
|- | |- | ||
| || | | 2 | ||
| 85960 | |||
| 100.872 | |||
| 8670951 | |||
| 175 | |||
| 8670776 | |||
|- | |- | ||
| | | 5 | ||
| 93351 | |||
| 168.424 | |||
| 15722556 | |||
| 194 | |||
| 15722362 | |||
|- | |- | ||
| || | | | ||
|} | |||
'''PCR Results with Corrected COncentration Values''' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''PCR Tube''' | |||
| align="center" style="background:#f0f0f0;"|'''Average INTDENS Value''' | |||
| align="center" style="background:#f0f0f0;"|'''PCR Product Concentration (µL/mL)''' | |||
| align="center" style="background:#f0f0f0;"|'''Corrected PCR Product Concentration (µL/mL)''' | |||
|- | |- | ||
| || | | P1A||2744551||-0.127724333||-1.532692 | ||
|- | |- | ||
| || | | P1B||2653457||-0.173271667||-2.07926 | ||
|- | |- | ||
| | | P1C||2690172||-0.154914||-1.858968 | ||
|- | |- | ||
| || | | +C+ ||18123980||7.561990167||90.743882 | ||
|- | |- | ||
| ||0. | | P2A||2786881||-0.106559667||-1.278716 | ||
|- | |- | ||
| || | | P2B||3208832||0.104416||1.252992 | ||
|- | |- | ||
| || | | P2C||1965640||-0.51718||-6.20616 | ||
|- | |- | ||
| || | | -C- ||2538375||-0.230812667||-2.769752 | ||
|- | |- | ||
| | | | ||
|} | |} | ||
'''Fitting a Straight Line'''<br> | '''Fitting a Straight Line'''<br> | ||
Line 193: | Line 336: | ||
'''PCR Results Summary'''<br> | '''PCR Results Summary'''<br> | ||
A Polymerase Chain Reaction (PCR) was completed for 8 DNA samples--3 DNA samples from each of two patients and a positive and a negative control sample. After the reactions and | A Polymerase Chain Reaction (PCR) was completed for 8 DNA samples--3 DNA samples from each of two patients and a positive and a negative control sample. After the reactions proceeded and cooled down, the samples were tested on a fluorimeter for amplification of DNA using SYBR Green I as a stain. The controls were necessary to ensure amplification results for the patients' samples had a comparative standard and the results were due to the procedure and not an external affect. Using a known concentration of DNA from the calibration phase, a standard curve was created that was a basis for conversion from imageJ INTEN values to DNA concentrations for the PCR samples. <br> | ||
Your positive control PCR result was 90.744 μg/mL <br> | Your positive control PCR result was 90.744 μg/mL <br> | ||
Your negative control PCR result was -2.770 μg/mL <br> | Your negative control PCR result was -2.770 μg/mL <br> | ||
Patient | Patient 1, ID: 51917 samples hardly showed any visibly show green <br> | ||
Patient | Patient 2, ID: 17539 all of their samples showed little to no green dye. <br> | ||
Patient 1, ID: 51917 samples were negative, were a bit blue but had little to no green, and would therefore also be considered negative for the diseased DNA. The average DNA concentration for Patient 1 was -1.824 μg/mL. <br> | |||
Patient | Patient 2, ID: 17539 samples were mostly negative, with an average concentration of -2.077, but one of the samples showed a bit of green with a calculated DNA concentration much farther from the negative control than the other results, but still farther from the positive control. The test would be considered negative for the diseased DNA, but with the one sample in disagreement with the other two, a retest may be desired to ensure that the patient is negative for the diseased DNA. | ||
<br> | <br> |
Latest revision as of 08:54, 17 April 2014
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OUR TEAM
LAB 5 WRITE-UPBackground InformationSYBR Green Dye
The way the fluorescence works is by utilizing a green dye that activates only under the presence of double stranded DNA (or dsDNA).
ProcedureSmart Phone Camera Settings
The camera phone was set up in the cradle with an angle to view the side of the drop. The fluorimeter was raised about 1 1/4 inches to accomodate for the phone height.
The phone was removed from this image to simplify and completely show distance setup.
Solutions Used for Calibration
1. Turn on the flourimeter. Place the super hydrophobic slide on the flourimeter. On the hydrophobic side of the flourimeter, place a 80 microliter drop of SYBR GREEN I on the middle rows of the slide. 2. Now on the sphere of dye that is on the slide, add 80 microliter of one of the solutions being tested. This will now react or not. 3. Next the flourimeter will shine a blue light, align the slide so that the drop sample is in the middle of this light. 4. Acquire a box that can cover the flourimeter in order to create a dark environment. Place a smartphone in the cradle, and use a timer on the camera to take a picture of the drop. Once the timer starts quickly put the box over the whole setup, so that outside light does not interfere.
Data AnalysisRepresentative Images of Samples High DNA - using green channel
Image J Values for All Samples
PCR Results with Corrected COncentration Values
Intends values for each Calf Thymus DNA cocnentration. PCR Results Summary A Polymerase Chain Reaction (PCR) was completed for 8 DNA samples--3 DNA samples from each of two patients and a positive and a negative control sample. After the reactions proceeded and cooled down, the samples were tested on a fluorimeter for amplification of DNA using SYBR Green I as a stain. The controls were necessary to ensure amplification results for the patients' samples had a comparative standard and the results were due to the procedure and not an external affect. Using a known concentration of DNA from the calibration phase, a standard curve was created that was a basis for conversion from imageJ INTEN values to DNA concentrations for the PCR samples.
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