Difference between revisions of "Assignment 10 Overview"
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=Assignment 10= | =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. | + | 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: |
− | * | + | * Collecting data |
− | * | + | * Analyzing data, and |
− | * | + | * Identifying your unknown sample and discussing your results. |
==In preparation== | ==In preparation== | ||
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==Collect Data== | ==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. | + | 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: | When you're ready, (and if you haven't already done so) choose which of the following axes you'd like to explore: | ||
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{{Template:Biohazard warning|message=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 <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}} | {{Template:Biohazard warning|message=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 <code>../EHS Guidelines/MSDS Repository</code> 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. | ||
{{Template: Assignment Turn In|message = 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.}} | {{Template: Assignment Turn In|message = 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.}} | ||
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− | |||
* Acquire two or (preferably) three melting curves for each known and unknown sample. | * Acquire two or (preferably) three melting curves for each known and unknown sample. | ||
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** 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? | ** 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? | ||
− | {{Template:Environmental Warning|message=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.}} | + | {{Template:Environmental Warning|message=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.}} |
+ | |||
+ | {{Template:Assignment Turn In|message= 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 using beater DNA, which is the same as sample A, but prepared with SYBR green. Which dye would you use if you wanted to make the most accurate measurement of ''T<sub>m</sub>''? Why? | ||
+ | }} | ||
{{Template:Assignment Turn In|message = | {{Template:Assignment Turn In|message = | ||
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{{Template:Assignment Turn In|message=<br> | {{Template:Assignment Turn In|message=<br> | ||
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* Document the regression model you used to analyze your data | * Document the regression model you used to analyze your data | ||
** See [[Assignment 9, Part 1: model function]] | ** See [[Assignment 9, Part 1: model function]] | ||
** Explain the model parameters using bullet points or in a table. | ** Explain the model parameters using bullet points or in a table. | ||
* Plot <math>V_{f,measured}</math> and <math>V_{f,model}</math> versus <math>T_{block}</math> for a typical run of each samples type. Use the smallest number of axes that clearly conveys the data. | * Plot <math>V_{f,measured}</math> and <math>V_{f,model}</math> versus <math>T_{block}</math> 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 | + | * 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, ([http://measurebiology.org/wiki/File:Residual_plot_for_DNA_data.png example plot]). | * For a typical curve, plot residuals versus time, temperature, and fluorescence, ([http://measurebiology.org/wiki/File:Residual_plot_for_DNA_data.png example plot]). | ||
* For at least one experimental trial, plot <math>\text{DnaFraction}_{inverse-model}</math> versus <math>T_{sample}</math> ([http://measurebiology.org/wiki/File:Inverse_cuvrve.png example plot]). On the same set of axes plot DnaFraction versus <math>T_{sample}</math> 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. | * For at least one experimental trial, plot <math>\text{DnaFraction}_{inverse-model}</math> versus <math>T_{sample}</math> ([http://measurebiology.org/wiki/File:Inverse_cuvrve.png example plot]). On the same set of axes plot DnaFraction versus <math>T_{sample}</math> 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. | ||
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#* Quantify the confidence you have in your result. | #* Quantify the confidence you have in your result. | ||
#* How do your estimates for <math>\Delta H, \Delta S</math> and ''T<sub>m</sub>'' compare to the predicted values from DINAmelt (or your favorite software)? | #* How do your estimates for <math>\Delta H, \Delta S</math> and ''T<sub>m</sub>'' compare to the predicted values from DINAmelt (or your favorite software)? | ||
− | |||
# Discussion: | # Discussion: | ||
#* Discuss the validity of assumptions in the regression model. | #* Discuss the validity of assumptions in the regression model. |
Revision as of 16:53, 13 November 2017
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:
- Collecting data
- Analyzing data, and
- Identifying your unknown sample and discussing your results.
In preparation
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
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.
- 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?
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).
|
Results
Resources
Background reading
- Electronics primer
- Real electronics
- Impedance analysis
- Transfer functions and Bode plots
- Input and output impedance
- DNA melting: Identifying the unknown sample
- DNA Melting Thermodynamics
- DNA Melting: DNA Sequences
- DINAMelt Web Server
Code examples and simulations
- DNA Melting: Simulating DNA Melting - Basics
- DNA Melting: Simulating DNA Melting - Intermediate Topics
- DNA Melting: Model function and parameter estimation by nonlinear regression
Subset of datasheets
(Many more can be found online or on the course share)