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. This assignment has 3 main parts: | + | 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. [http://measurebiology.org/wiki/DNA_Melting:_DNA_Sequences This page] lists the DNA sequences of your known samples. This assignment has 3 main parts: |
# collecting data, | # collecting data, | ||
# analyzing data, | # analyzing data, | ||
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{{Template:Assignment Turn In|message=<br> | {{Template:Assignment Turn In|message=<br> | ||
− | '''Individually,''' | + | '''Individually,''' (37/100 points) |
* 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. | ||
+ | ** Include all equations incorporated into the model and the linear regression. | ||
* 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 uncertainties for each experimental run. Also include the estimated melting temperature for each run. | + | * Provide a table of the best-fit model parameters for <math> \Delta H</math> and <math>\Delta S</math> and their 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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{{Template:Assignment Turn In|message= | {{Template:Assignment Turn In|message= | ||
'''As a team,''' | '''As a team,''' | ||
− | # Results: | + | # Results: (20/100 points) |
#* Identify your unknown sample (or state that your investigation did not provide a conclusive answer). | #* Identify your unknown sample (or state that your investigation did not provide a conclusive answer). | ||
#* 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)? | ||
#* How do your estimates for <math>\Delta H, \Delta S</math> and ''T<sub>m</sub>'' compare to results from other groups and/or instructor data? | #* How do your estimates for <math>\Delta H, \Delta S</math> and ''T<sub>m</sub>'' compare to results from other groups and/or instructor data? | ||
− | # Discussion: | + | # Discussion: (29/100 points) |
#* 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 ''T<sub>m</sub>''? Why? | #* 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 ''T<sub>m</sub>''? Why? | ||
#* Discuss the validity of assumptions in the regression model. | #* Discuss the validity of assumptions in the regression model. |
Latest revision as of 16:37, 8 May 2018
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 page lists the DNA sequences of your known samples. This assignment has 3 main parts:
- collecting data,
- analyzing data,
- identifying your unknown sample and discussing your results.
In preparation
Individually, (1/100 points) 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 & Disscussion
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)