User:Justinhlo

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Instructions: Copy this page to your user page and fill it out on the wiki for submission. 
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==DNA Melting Project: IAP 2007==
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==Record for experiment addressing Coli-roid operating conditions==
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===Ionic Strength===
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===Lab section===
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Using either NaCl or KCl (is there an advantage to one over the other?  Does Na<sup>+</sup> or K<sup>+</sup> interact more with O<sup>-</sup>?).  Original 19-bp sequence is fine ..
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''Choose one''
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{|
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Goal: show how the set-up can be used to investigate the significant effects of ionic particle concentrations on DNA melting parameters.
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| style="width:100px" | <b>((T/R))</b>
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| style="width:100px" | W/F
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|}
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===Team color===
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Control and Experimental Groups:
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''Choose one''
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0 mM (control),
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1 mM,
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10 mM (original module condition),
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150 mM (physiological conditions, see http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2703503&dopt=Abstract),
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1000 mM (SantaLucia paper’s conditions – I’m curious to see if we get similar results).
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{|
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These conditions form an approximately logarithmic distribution between 0 M and 1 M.  This is justified because the projected dependence of ΔS and ΔH on ionic concentration is also logarithmic.  The 150 mM may be replaced by 100 mM if it is deemed more desirable. 
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| style="width:100px" | Green
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| style="width:100px" | <b>((Purple))</b>
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| style="width:100px" | Blue
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| style="width:100px" | Red
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| style="width:100px" | Pink
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| style="width:100px" | Yellow
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===Describe the experimental variations you are comparing.===
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It may be worth looking into the 50 mM concentration, because this is what PCRs are run at, and it is also what many of the models have actually been designed for.
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We are comparing the type of medium used to grow the E. Coli: LB vs. SOC (rich medium). 
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===Mismatches===
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===What were the:===
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C, T are pyrimidines (small)
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A, G are purines (large)
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[...]
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====Densities of your two samples?====
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===Length===
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[...]
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LB: 2.77x10<sup>9</sup> cells/mL<br>
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===Potential Models for DNA modeling===
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SOC: 1.53x10<sup>9</sup> cells/mL
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====β-gal values measured for the two samples?====
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This interesting paper actually lists a good number of methods (including the one we used in the module).  http://bioinformatics.oxfordjournals.org/cgi/content/full/21/6/711.  However, while it compares the methods thoroughly, it does not run the empirical experiments in order to see which one is actually right.
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LB: 7995 units<br>
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Here are a few models worth investigating:
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SOC: 6905 units
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The very basic equation used for very short sequences: Tm= (wA+xT) * 2 + (yG+zC) * 4.
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The standard G-C content equation: Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC).
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====Concentrations of total RNA that you isolated from the two samples?====
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The modified G-C content equation, with more length-dependent consideration: Tm = 100.5 + 41*(yG+zC-836.4)/(wA+xT+yG+zC).
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The NN model:  
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LB: 158.4 μg/mL<br>
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It seems that the last one here is probably the only one that actually tries to include entropic and enthalpic conditions.  That means we have limited options for the fitting of the actual curve.
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SOC: 46.4 μg/mL
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====Method of cDNA synthesis====
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''Choose one''
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{|
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| style="width:100px" | <b>((Epicenter))</b>
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| style="width:100px" | Invitrogen
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|}
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===Primer pair used===
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''Choose one''
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{|
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| style="width:200px" | 5' end of LacZ gene
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| style="width:200px" | Middle of LacZ gene
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| style="width:200px" | 3' end of LacZ
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|}
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===Wells for your samples:===
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[[Image:Macintosh HD-Users-nkuldell-Desktop-RTPCRplate.png| RT-PCR 96 well plate]]
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Revision as of 14:41, 8 January 2007

Contents

DNA Melting Project: IAP 2007

Ionic Strength

Using either NaCl or KCl (is there an advantage to one over the other? Does Na+ or K+ interact more with O-?). Original 19-bp sequence is fine ..

Goal: show how the set-up can be used to investigate the significant effects of ionic particle concentrations on DNA melting parameters.

Control and Experimental Groups: 0 mM (control), 1 mM, 10 mM (original module condition), 150 mM (physiological conditions, see http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2703503&dopt=Abstract), 1000 mM (SantaLucia paper’s conditions – I’m curious to see if we get similar results).

These conditions form an approximately logarithmic distribution between 0 M and 1 M. This is justified because the projected dependence of ΔS and ΔH on ionic concentration is also logarithmic. The 150 mM may be replaced by 100 mM if it is deemed more desirable.

It may be worth looking into the 50 mM concentration, because this is what PCRs are run at, and it is also what many of the models have actually been designed for.

Mismatches

C, T are pyrimidines (small) A, G are purines (large) [...]

Length

[...]

Potential Models for DNA modeling

This interesting paper actually lists a good number of methods (including the one we used in the module). http://bioinformatics.oxfordjournals.org/cgi/content/full/21/6/711. However, while it compares the methods thoroughly, it does not run the empirical experiments in order to see which one is actually right.

Here are a few models worth investigating: The very basic equation used for very short sequences: Tm= (wA+xT) * 2 + (yG+zC) * 4. The standard G-C content equation: Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC). The modified G-C content equation, with more length-dependent consideration: Tm = 100.5 + 41*(yG+zC-836.4)/(wA+xT+yG+zC). The NN model: It seems that the last one here is probably the only one that actually tries to include entropic and enthalpic conditions. That means we have limited options for the fitting of the actual curve.

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