Assignment 7, Part 3: testing your instrument and measuring a DNA melting curve

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20.309: Biological Instrumentation and Measurement

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This is Part 3 of Assignment 7.

Assignment 7 measurements

Test your instrument with fluorescein

Use ~ 30μM fluorescein to test your instrument before you get started with DNA samples. Fluorescein is good for testing; it's cheap, non-toxic, and it does not bleach as quickly like LC Green.

  1. Pipette 500 μL of fluorescein into a glass sample vial.
  2. Pipette 500 μL of deionized water into a another sample vial.
  3. Alternate between the two samples. You should see a difference in the transimpedance amplifier output.

Measure signal to noise ratio

Measure the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 μL of 30 μM fluorescein and a vial containing the same amount of water.

  1. Run the Basic DNA Melter GUI.
  2. Place a vial of deionized water in your instrument.
  3. Clear the data and wait 10 seconds.
  4. Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data.
    • Be sure that all other conditions, such as temperature, are stable throughout the test.

Compute the signal to noise ratio, $ \text{SNR}=\frac{\langle V_{fluorescein} \rangle - \langle V_{water} \rangle}{\sigma_{fluorescein} } $, where $ V_{fluorescein} $ is the portion of the data recorded with a fluorescein sample, $ V_{water} $ is the portion of the signal corresponding to the water sample, and $ \sigma_{fluorescein} $ is the standard deviation of $ V_{fluorescein} $.


Pencil.png Report your signal-to-noise measurement.


Make a DNA melting curve

Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for a 20 bp DNA and green dye solution.


Biohazard.jpg LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under ../EHS Guidelines/MSDS Repository in the course locker for more information.


  1. Pipet 500 μL of DNA plus dye solution into a glass vial.
  2. Pipet up to 20 μL of mineral oil on top of the sample to help prevent evaporation.
    • The oil layer will reduce evaporation.
    • Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal.
    • Keep the sample vertical to make sure the oil stays on top.
  3. Make sure that the Diablotek power supply is off and the sample block is near room temperature.
  4. Place the vial in the instrument
    • To reduce bleaching, keep the LED off until you start the measurement.
  5. Open and run the Basic DNA Melter GUI and follow the instructions there.
  6. Confirm that sample block temperature displayed in Basic DNA Melter GUI is near room temperature.
  7. Turn on the Diablotek power supply until the block temperature reaches 95°C.
  8. Attempt to hold the temperature at 95°C for a minute or so by repeatedly switching the Diablotek power supply off and on.
  9. Switch off the power supply and allow the block to cool down to room temperature.
    • Be sure to save your data so you can plot it in your report.

You can use the same sample for several heating/cooling cycles.
Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low.

To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.


Global Tree.gif Discard pipette tips with DNA sample residue in the pipette tips container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.



Pencil.png
  1. Plot at least one melting curve.
    • The plot should have temperature in °C on the horizontal axis and fraction of double stranded DNA on the vertical axis.
    • On the same set of axes, include your simulated curve from the pre-lab problem B,2 generated by DINAMelt, OligoCalc, or another software simulator.
    • Also on the same set of axes, plot the output of the DnaFraction function evaluated with best-fit parameters. You may use nlinfit to choose the best-fit parameters or you may choose them manually. (See: DNA Melting: Simulating DNA Melting - Basics)
    • Include a legend.
  2. Report the estimated melting temperature and the best-fit values of ΔH°, ΔS°.


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