Difference between revisions of "Assignment 7, Part 3: testing your instrument and measuring a DNA melting curve"
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Compute the signal to noise ratio, <math>\text{SNR}=\frac{\langle V_{fluorescein} \rangle - \langle V_{water} \rangle}{\sigma_{fluorescein} }</math>, where <math>V_{fluorescein}</math> is the portion of the data recorded with a fluorescein sample, <math>V_{water}</math> is the portion of the signal corresponding to the water sample, and <math>\sigma_{fluorescein}</math> is the standard deviation of <math>V_{fluorescein}</math>. | Compute the signal to noise ratio, <math>\text{SNR}=\frac{\langle V_{fluorescein} \rangle - \langle V_{water} \rangle}{\sigma_{fluorescein} }</math>, where <math>V_{fluorescein}</math> is the portion of the data recorded with a fluorescein sample, <math>V_{water}</math> is the portion of the signal corresponding to the water sample, and <math>\sigma_{fluorescein}</math> is the standard deviation of <math>V_{fluorescein}</math>. | ||
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Revision as of 19:49, 26 October 2017
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.
- Pipette 500 μL of fluorescein into a glass sample vial.
- Pipette 500 μL of deionized water into a another sample vial.
- 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.
- Run the
Basic DNA Melter GUI
. - Place a vial of deionized water in your instrument.
- Clear the data and wait 10 seconds.
- 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} $.
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.
- Pipet 500 μL of DNA plus dye solution into a glass vial.
- 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.
- Make sure that the Diablotek power supply is off and the sample block is near room temperature.
- Place the vial in the instrument
- To reduce bleaching, keep the LED off until you start the measurement.
- Open and run the Basic DNA Melter GUI and follow the instructions there.
- Confirm that sample block temperature displayed in Basic DNA Melter GUI is near room temperature.
- Turn on the Diablotek power supply until the block temperature reaches 95°C.
- Attempt to hold the temperature at 95°C for a minute or so by repeatedly switching the Diablotek power supply off and on.
- 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.
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- Overview
- Part 1: Pre-lab problems
- Part 2: Build an instrument to measure temperature and fluorescence
- Part 3: Testing and measuring stuff