Our procedure went smoothly when pipetting. The pre-lab was helpful for our group. It gave clear instructions how to correctly use a micropipette which helped move our lab on smoothly. The first stop on the micropipette is used initially to gather the fluid and the second stop is used when distributing the fluids. The final reactions should have exactly the same amount of liquid in each tube. There may be slight discrepancies due to air being trapped in the micropipette or error in pipetting procedure. There was a small amount of liquid left at the bottom of some of the tubes. This could come from error in pipetting or initial measurement of the samples. The labeling scheme did not need to be changed from what was originally proposed in lab write up four.
Fluorimeter Procedure
Imaging set-up First a phone is propped up so that it can take pictures. The fluorimeter is elevated so that it is level with the camera. The phone is then moved about 5cm away from the fluorimeter. Once the picture looks to be in focus, the camera is likely in a good position. A super hydrophobic piece of glass is set inside the fluorimeter.
Placing Samples onto the Fluorimeter
Using a micropipette, insert 80 microliters seperatly of the different concentrates of Calf Thymus DNA (5, 2, 1, 0.5, 0.25, 0) onto the hydropobic glass plate between two center dots
Using a micropipette with a new tip, add 80 microliters of SYBR Green into the Calf Thymus DNA droplet, this will help show that the calibration is correct
Using a micropipette with a new tip, insert 100 microliters of each different original templates of DNA into different the different test tubes with buffer
Using a micropipette with a new tip, add 80 microliters of SYBR Green is placed on the hydrophobic glass piece in between two center dots
Using a micropipette with a new tip, add 80 microliters of the prepared DNA into the SYBR Green droplet
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was 14.683167 μg/mL
Our negative control PCR result was -0.51816945 μg/mL
Observed results
Patient 37987 : This patient's sample had virtually no green in the droplet. The PCR product concentration was around -2.47428645 μg/mL.
Patient 29674 : This patient's sample had a very small amount of green dye in it, but was still almost completely clear. The PCR product concentration was around -2.043723 μg/mL.
Conclusions
Patient 37987 : It is highly likely that this patient is negative. This is do to how similar the initial PCR product concentration was to the negative control and to the fact that there was almost no green dye whatsoever in the pictures.
Patient 29674 : It is also highly likely that this patient is negative. This is do to how similar the initial PCR product concentration was to the negative control and to the fact that there was very little green dye in the pictures.
BME 100 Spring 2018
