LAB 5 WRITE-UP
PCR Reaction Report
We were given a micropipettor, empty tips, 8 DNA primer mixes, 8 PCR reaction mix (MgCl2, Taq DNA Polymerase, dNTP's), a strip of empty PCR tubes, a beaker for used tip disposal, and an openPCR machine that is shared between two groups. The 8 empty PCR tubes were labeled based on labels created in Part A of lab. Using the micropipettor, Evan and Noah transferred 50 μl of PCR reaction mix into an empty PCR tube. Then after discarding the tip and placing a new tip, they transferred 50 μl of positive DNA primer mix to the same tube. This was repeated for the negative control, patient 1 replicates 1,2, and 3, and patient 2 replicates 1,2, and 3. The lids were then closed, taken to the openPCR machine, and with the help of the TA were placed into the openPCR machine and programmed according to the procedure on page 6 of the Lab Workbook. Noah and Evan were pretty good at micropipette technique and enjoyed doing it while Jeremy and I watched to make sure the correct mixes were being placed in the correct tubes and that each tip was discarded after use and a new tip placed on the micropipette.
Picture of micropipetting in Lab 5.
The pre-lab reading helped us to understand how the PCR reaction lab is conducted and the videos on how to use the micropipette were vital to doing the lab properly. For the micropipette, the first stop sucks liquid into the tip and the second stop releases the liquid. Overall our group did well in the PCR reaction lab, from using the micropipette to handling the openPCR machine.
- In order to be able to take photos from the proper angle, you must first place the fluorimiter on an elevated surface that would be on the level of your phone camera.
- Set your phone up on the provided stand and ensure that the camera is in line with where the drops are going to be placed, and move it as close as possible to the drop.
- Be sure to have a timer on your camera so that when the light box is shut, you won't have to worry about taking the individual pictures.
- Make sure the blue LED light is in line with where the drops are going to be placed.
- Once placing the drop on the slide, start the timer on the camera and close the light box over the fluorimeter and take a total of 3 pictures.
- Remove the box, remove the drop and repeat the procedure with the rest of the samples.
Placing Samples onto the Fluorimeter
Fluorimeter setup with droplet
- The first two droplets will be placed on the first two dots in the middle of the slide.
- Make sure the light of the flourimeter is shining in between the first and second dot of the slide.
- With a clean pipette tip, take 80 microliters of SYBR GREEN and place the drop on the first dot.
- Eject the tip into the waste cup.
- With another clean tip, take 80 microliters of a sample and place it on the second dot on the slide, just behind the first drop that you placed.
- When the two drops are placed, they should combine into one big drop, spanning the length between the two dots on the slide.
- Eject the tip into the waste cup and take photos of your sample in a light box.
- Set the micropipette to 160 microliters and pipette up the droplet from the slide and eject the tip, which contains the 160 microliters of your sample, into the waste cup.
- Move the slide back so that the light is shining in between the third and fourth dots of the slide.
- Repeat steps 3-7 until you have completed taking photos of all of your samples.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL sample
0.5 μg/mL sample
Calibrator Mean Values
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
PCR Results: Summary
- Our positive control PCR result was 2.2 μg/mL
- Our negative control PCR result was 1.5 μg/mL
- Patient 29596 : When we placed the replicates 1, 2, and 3 of patient 1 in the fluorimeter with the SYBR GREEN, we did not see any green fluorescence for any of the replicates, and this was due to the fact that the range of μg/mL of DNA in patient one ranged from -.05 μg/mL to .8 μg/mL, which resulted in no visible fluorescence.
- Patient 61164 : When we placed the replicates 1, 2, and 3 of patient 2 in the fluorimeter with the SYBR GREEN, we saw a strong and vibrant fluorescence, and this was due to the fact that the ranges of μg/mL fo DNA in patient 2 was around 2.29 μg/mL to 2.45 μg/mL.
- Patient 29596 : When compared to the positive control of 2.2 μg/mL and the negative control of 1.5 μg/mL, and also when looked at in the fluorimeter, it is clear that Patient 29596, with a value of -.05 μg/mL of DNA, is negative and that there is no evidence that there is any of the targeted DNA in the PCR sample.
- Patient 61164 : When compared to the positive control of 2.2 μg/mL and the negative control of 1.5 μg/mL, and also when looked at in the fluorimeter, it is clear that Patient 61164, with a value of 2.45 μg/mL of DNA, is positive and there is strong evidence that there is a presence of the targeted DNA in the PCR sample.