Our team learned the importance of micro pipetting while setting up our DNA samples for PCR. When doing this lab, we realized that the most important factor was labeling the tubes correctly. If the tubes weren't labeled according to their matching DNA, the samples could be contaminated. Before starting the micro pipetting process, we reviewed the pre-lab which was extremely helpful. The interactive videos allowed us to understand how to use the micropipettor and specified the order in which the process needed to be done. Due to the pre-lab, we were able to successfully complete the process without error. When transferring the PCR reaction mix into the empty tubes, it was important to press the pipettor to the first stop, then insert the tip into the liquid, and slowly release it. If it was placed in the liquid, then pressed to the first stop, it would have caused air bubbles. Then, to transfer it into the empty tube, we inserted the tip then pressed the stopper to the second stop to release the liquid. Additionally, we had to make sure to change the tip each time we used the pipettor to make sure there was no cross contamination. Since we knew the difference between the first and second stop, we were able to transfer all of the liquid from the PCR reaction mix and the DNA samples to their proper tubes without leaving any behind in the old tube.
Fluorimeter Procedure
Imaging set-up
In order to start the fluorimeter process, it was crucial to set up a smartphone to take pictures of the drops on the glass slide. To do this, we first decided which group member had the best quality camera, which was the iPhone 6+, and then placed it into the holder. To get a clear image of the drop, we put plastic containers under the phone holder to raise the lense so that it was level with the drop horizontally. After the height was adjusted, the distance was set. It was important to have the camera close enough to the drop but still be focused. After we tried different positions, we found that when the camera was at an ideal spot 5 cm from the platform. So, for each picture, we were careful in making sure that the camera was centered on the drop and was 5 cm away from the platform. This ensured that each picture was clear for the rest of the lab.
Placing Samples onto the Fluorimeter
Put on gloves and get a glass side and put the smooth side face down. Get the micropipettor and place a 80 microliter drop of SYBR GREEN I in the middle of the first two bars on the slide.
Now place a 80 microliter drop of the first calf thymus solution on the first drop (add to it). Turn on the blue LED light and make sure it is lined up with the drop so that the light is shining through it.
Set up the camera phone at least 4 cm away from the slide and focus the camera on the drop. Record the distance between the slide and the phone.
Set a timer on the phone so that you can close the box before the picture is taken. Take three separate pictures.
Micropipette the drop off of the slide and move it to the next two clean rows.
Repeat these steps for the rest of the calf thymus solutions.
Get the 8 PCR reaction samples (DNA) and the 8 tubes with the 500 microliters of buffer. Label each of the 8 tubes with the buffer to match each of the PCR reaction samples labels.
Set the micropipettor to 120 microliters, attach a disposable tip and then transfer all 100 microliters of the PCR reaction sample into its properly labeled buffer tube. Make sure to dispose the tip each time you transfer a new PCR reaction.
After transferring the PCR reaction sample into the buffer, invert the tube so that the two liquids mix properly.
Now repeat the same fluorimeter process used for the calf thymus for the 8 PCR and buffer solutions.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL Sample
0.5 μg/mL Sample
Zero DNA Sample
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL)
Final DNA concentration in SYBR Green I solution (µg/mL)
Sample Number
RAWINTDEN DROP - BACKGROUND
MEAN
Standard Deviation
Image 1
Image 2
Image 3
5
2.5
C-1
29120173
29082565
28957451
29053396.33
85192.25703
2
1
C-2
27322143
27293299
27328580
27314674
18789.00506
1
0.5
C-3
22521520
22447342
21565630
22178164
531765.0043
0.5
0.25
C-4
22465814
23205053
22822986
22831284.33
369689.3582
0.25
0.125
C-5
18959821
18332789
17866431
18386347
548659.0631
0
0
C-6
12806746
12805561
12643946
12752084.33
93652.41806
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive Control Sample
Negative Control Sample
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
MEAN (of RAWINTDEN DROP - BACKGROUND)
PCR Product Concentration (µg /mL)
Total Dilution
Initial PCR Product Concentration (µg /mL)
G6P
19686118
7.707955364
12
110.9945576
G6N
10667719.67
4.176867529
12
50.12241035
G61-1
10736477.67
4.203789221
12
60.53456537
G61-2
11832771.67
4.63303511
12
55.59642132
G61-3
11634644.33
4.554598
12
54.6655176
G62-1
11301837.33
4.425151656
12
53.10181987
G62-2
9006830
3.52655834
12
55.01431165
G62-3
12380880.33
4.847643042
12
58.17171651
PCR Results: Summary
Our positive control PCR result was 110.99 μg/mL
Our negative control PCR result was 50.122 μg/mL
Observed results
Patient 68474: There is a very clear difference from the positive constraint and Patient 68474. Patient 1 has has little to no green visiblity in the three images. Since the images and the concentrations 60.535, 55.596, 54.666μg/mL look closer to the negative control, it can be confirmed that this patient will test negative for the disease.
Patient 95650: There is a clear difference between the positive constraint and Patient 95650. Patient 2 additionally has little to no green in the three images. Since images and the following concentrations of 53.102, 55.014, 58.171μg/mL are closer in value to the negative control, it can be inferred that this patient will test negative for the disease.
Conclusions
Patient 68474: PCR test yields an average value of 56.932 which is closer to the negative control value of 50.122. The conclusion was drawn that the patient is negative for the diseased SNP.
Patient 95650 : PCR test yields an average value of 55.429 which is closer to the negative control value of 50.122. The conclusion was drawn that the patient is negative for the diseased SNP.
BME 100 Spring 2018
