Assignment 10 Overview

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20.309: Biological Instrumentation and Measurement

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Assignment 10

In the final assignment of the DNA melting lab, you will measure DNA melting curves for 3 known samples and one unknown. For each sample you will extract the best-fit thermodynamic parameters, and use them to identify your unknown sample. This assignment has 3 main parts:

  1. collecting data,
  2. analyzing data,
  3. identifying your unknown sample and discussing your results.

In preparation


Pencil.png Compose an entertaining, exhilarating, thought-provoking, or melancholy Haiku on the subject of DNA melting.


Collect Data

You may want to check your instrument to make sure it is still working and reliable. Use fluorescein and beater DNA to test until you are satisfied with the results. The DNA samples you will receive for the next part of the lab are prepared with LC green fluorescent dye, as opposed to SYBR green dye which you've been using so far. LC green is somewhat dimmer than SYBR green, so you may need to tweak your system accordingly.

When you're ready, (and if you haven't already done so) choose which of the following axes you'd like to explore:

  • DNA length
  • Number of mismatches
  • Salt concentration


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.


You will receive 1.5 mL each of four samples. Three of the samples will be identified by their sequence, salt ion concentration, and degree of complementarity (see DNA_Melting:_DNA_Sequences for sequence details and the sample naming key). The fourth sample matches one of the three identified samples. You will not be told which one.


Pencil.png Document your data collection procedure. Report instrument settings for each trial, including control software parameters. Refer to the lab manual wiki pages when appropriate, and describe any changes you made.


  • Acquire two or (preferably) three melting curves for each known and unknown sample.
    • Running the samples more than once will provide more confidence in your result.
    • It may be wise to analyze the data as you go. Ask yourself: is the melting temperature approximately what I would expect? Do the trends in melting temperature for the known samples agree with my intuition?


Global Tree.gif Discard pipette tips with DNA sample residue in the pipette tips or the Biohazard Sharps 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, or aspirate them into the biowaste flask to the left of the sink.



Pencil.png Plot all of your raw data (as fluorescence vs. block temperature) on the smallest number of axes that clearly conveys the dataset. Include only data generated by your own group.
  • Data from the many sample runs overlaps, which makes presenting so much data on a small number of axes a real challenge.
  • Devise a combination of line colors, line thicknesses, and marker symbols that produces clear plot. If two sample types have a great deal of overlap, there may be no choice but to plot them on separate axes.
  • One approach that works well for some datasets is to plot a subsampled version of each trial using discrete markers. Vary the color and form to differentiate between sample types and individual trials.


Analyze data

Use your code developed in Assignment 9 to fit your data to the mechanistic model discussed in lecture (an in Assignment 9, Part 1: model function).


Pencil.png
  • Document the regression model you used to analyze your data
  • Plot $ V_{f,measured} $ and $ V_{f,model} $ versus $ T_{block} $ for a typical run of each samples type. Use the smallest number of axes that clearly conveys the data.
  • Provide a table of the best-fit model parameters and uncertainties for each experimental run. Also include the estimated melting temperature for each run.
  • For a typical curve, plot residuals versus time, temperature, and fluorescence, (example plot).
  • For at least one experimental trial, plot $ \text{DnaFraction}_{inverse-model} $ versus $ T_{sample} $ (example plot). On the same set of axes plot DnaFraction versus $ T_{sample} $ using the best-fit values of ΔH and ΔS. Finally, plot simulated dsDNA fraction vs. temperature using data from DINAmelt or another melting curve simulator.


Results & Disscussion


Pencil.png
  1. Results:
    • Identify your unknown sample (or state that your investigation did not provide a conclusive answer).
    • Quantify the confidence you have in your result.
    • How do your estimates for $ \Delta H, \Delta S $ and Tm compare to the predicted values from DINAmelt (or your favorite software)?
    • How do your estimates for $ \Delta H, \Delta S $ and Tm compare to results from other groups and/or instructor data?
  2. Discussion:
    • LC green fluorescent dye saturates the binding sites of the double-stranded DNA. SYBR green, in turn, is non-saturating. Compare the melting temperatures that you measured for Sample A (20 bp, complete match, 100mM salt) to a measurement you made previously using the 'beater DNA'. The beater DNA is the same sequence and salt concentration as sample A, but prepared with SYBR green. Which dye would you use if you wanted to make the most accurate measurement of Tm? Why?
    • Discuss the validity of assumptions in the regression model.
    • Discuss any atypical results or data you rejected.
    • Discuss significant error sources.
      • Consider the entire system: the oligos, dye, the experimental method, and analysis methodology, and any other relevant factors.
      • Indicate whether each source likely caused a systematic or random distortion in the data.
      • Present error sources, error type and their resultant uncertainty on your data and results in a table, if you like.
    • Discuss additional unimplemented changes that might improve your instrument or analysis.


Back to 20.309 main page

Resources

Background reading

Code examples and simulations

Subset of datasheets

(Many more can be found online or on the course share)

  1. National Instruments USB-6212 user manual
  2. National Instruments USB-6341 user manual
  3. LF411 Op-amp datasheet
  4. LM741 Op-amp datasheet


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