The pre lab reading and online tutorial was really helpful in learning how to use a micropipette. The first stop on the micropipette is used to suck up the fluid and the second stop is used to eject all the fluid. The second stop is very important because it gives the sample a push of air, and that’s what makes micropipetting accurate in transferring small amounts of fluid. The final reactions in our experiment did have the same amount of liquid, all the liquid was transferred and there was no change in labeling. Our entire group all was able to micropipette, making the process easy for the group.
Fluorimeter Procedure
Imaging set-up
To capture accurate images of the DNA samples with the SYBR Green 1, we used an Iphone 6. To get the fluorimeter to the correct height, a box of micropipette tips was placed under it. To hold the phone still while taking a picture in the dark box, a picture timer was used, and the Iphone was sitting in a stand. We were able to focus the image by tapping on the screen. After pressing the capture button, we closed the box completely so that we could capture the picture with the most fluorescence.
Placing Samples onto the Fluorimeter
Take a slide out of the container and slide it into the flourimeter with the smooth side down.
Using a micropipettor, pipette 80 ul of SYBR Green 1 onto the slide in between the first and second divets with the drop between the light.
Using the same micropipette with a different tip, pipette 80 ul of calibration 5 onto the same drop of liquid as done in step two.
With the apparatus step up, focus the camera on the drop in the dark box. Touch the capture button and close the box completely.
Repeat steps 1-4 until all of the calibration pictures have been taken and until all of our DNA IDs have been used.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
DotPlotCurve
Above is the ImageJ image for the 5 (High) μg/mL calibrated sample.
DotPlotCurve
Above is the ImageJ image for the .5 (Low) μg/mL calibrated sample.
DotPlotCurve
Above is the ImageJ image for the 0 μg/mL calibrated sample.
Calibrator Mean Values
[ ] Calf Thymus DNA Solution (micrograms/mL)
MEAN RAWINTDEN DROP - BACKGROUND
STANDARD DEVIATION
5
11371352
657993.6478
2
12313197.3
962041.7177
1
8516698.33
236028.4715
0.5
3521823
338105.4879
0.25
6099816
436979.831
0
1973312
276091.563
Calibration curves
DotPlotCurve DotPlotCurve
Images of Our PCR Negative and Positive Controls
DotPlotCurve
This image is an example of the negative control of Calf Thymus DNA (In micrograms per mL).
DotPlotCurve
This image is an example of the positive control of Calf Thymus DNA (In micrograms per mL).
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
MEAN (RAWINTDEN DROP - BACKGROUND)
PCR Product Concentration (µg /mL)**
Total Dilution
Initial PCR Product Concentration (µg /mL)
+
398964.6667
-0.520207067
12
-6.2424848
-
158429.3333
-0.568314133
12
-6.8197696
1-1
382612.6667
-0.523477467
12
-6.2817296
1-2
441593.3333
-0.511681333
12
-6.140176
1-3
491605
-0.501679
12
-6.020148
2-1
130981.3333
-0.573803733
12
-6.8856448
2-2
124748
-0.5750504
12
-6.9006048
2-3
136689.6667
-0.572662067
12
-6.8719448
'**'Note: There was an unknown error in the derivation of the PCR Concentration which resulted in a negative concentration. This does not, however, effect the correlation and results of the data.
PCR Results: Summary
Our positive control PCR result was -0.520207067 μg/mL
Our negative control PCR result was -0.568314133 μg/mL
Observed results
Patient 16717: The direct images of this patient's DNA mixture were visibly green in the experimental set-up. It is also noteworthy that the PCR Product concentrations for this patient were: -0.523477467, -0.511681333, and -0.501679, all in units of μg/mL.
Patient 11408: The images of this patient's DNA mixture were relatively lacking in fluorescence, and had no visible green color associated with the solution. Quantitatively, the PCR Product concentrations for this patient were -0.573803733, -0.5750504, and -0.572662067 (μg/mL).
Conclusions
Patient 16717: From the results, it can be seen that the average value of the PCR Product concentration for this patient was -0.512279267 μg/mL which can be compared to the two controls. With respect to the negative control concentration, this average is closest to the positive control value of -0.520207067 μg/mL. For this reason, it can be accurately stated that this patient is positive for the disease marker.
Patient 11408: Looking at the three values of Patient 11408's PCR Product concentration, the average can be calculated to be -0.573838733 μg/mL. The negative control threshold for the disease marker was determined to be -0.568314133 μg/mL. By Comparing these two values, while keeping the positive control threshold in consideration, it can be seen that the two values are close, and it can be determined that this patient is negative for the disease marker.
Gel Electrophoresis Extra Credit Lab:
Number
Description
1
Ladder
2
Positive
3
Negative
4
1-1
5
1-2
6
1-3
7
2-1
8
2-2
9
2-3
Did the pre-lab reading help you? I have had experience twice with using the gel electrophoresis so I was fairly comfortable with the procedure; however, the pre-lab helped as a nice refresher.
Did you understand the difference between the first and second stop on the pipettor? The first stop is to pick up the sample; the second stop is to release the sample within the pipette tip.
Did the final reactions have exactly the same amount of liquid? For the most part the final reactions were close to one another with regards to the amount of liquids that they had.
Was there any liquid left in the tubes that the DNA samples and PCR reaction mix? Yes, there was some liquids left in the tubes that had the DNA samples and PCR reactions. This is due to the fact that some of the liquid was on the sides or sometimes the pipette didn’t pick up all the liquid despite our best efforts.
Did you have to change your labeling scheme? We did not have to change our labeling scheme - we just copied the label order from the sample tubes.
BME 100 Spring 2017
