Difference between revisions of "Assignment 10 Overview"
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'''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. |
Revision as of 18:33, 29 January 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 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).
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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)