User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/14: Difference between revisions

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{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:Logo_light_minimal_full_res.jpg|150px]]<span style="font-size:22px;"> WiFi coli: A communicolight system</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==PCR day==
==PCR day==
<br/ >
<br/ >
* Today, I wanted to do two PCRs: one in order to change the RBS of the luciferase as described in the [http://partsregistry.org/Part:BBa_K216015:Design| parts registry], and another to conduct a punctual mutation for the emission espectrum of luciferase.  
* Today, I wanted to do two PCRs: one in order to change the RBS of the luciferase as described in the [http://partsregistry.org/Part:BBa_K216015:Design parts registry], and another to conduct a punctual mutation for the emission spectrum of luciferase.  
<br/ >
<br/ >
* The first assay failed, as I missed the hot start step. The reaction was done as follows for 6.5 reactions:
* The first assay failed, as I missed the hot start step. The reaction was done as follows for 6.5 reactions using as a forward primer the rbs + 1 primer (syntethized to avoid the complications described in the parts registry above), and a dilution of DNA (5μL / 20 of H<sub>2</sub>O):
<br/ >
* The Fwd primer sequence is: 5' -> 3' GCT CTA GAG AGG AAA CAG CTA TGG AAG ACG CCA AAA AC
<br/ >
<br/ >
--> Mix 1 for 30 μL <--
--> Mix 1 for 30 μL <--
Line 34: Line 36:
<br/ >
<br/ >
* The 30 cycles were programed as follows:
* The 30 cycles were programed as follows:
<br/ >
<br/ >
- Initialization step: 95°C for 5 min. (only the 1st mix)<br/ >


- Initialization step: 95°C for 5 min. (only the 1st mix)<br/ >
- Hot start: Stop to add the second mix<br/ >
- Hot start: Stop to add the second mix<br/ >
- Denaturation step: 95°C for 45 seg. <br/ >
- Denaturation step: 95°C for 45 seg. <br/ >
- Annealing step: 60°C for 45 seg. <br/ >
- Annealing step: 60°C for 45 seg. <br/ >
- Extension/elongation step: 72°C for 1:50 min.<br/ >
- Extension/elongation step: 72°C for 1:50 min.<br/ >
- Final elongation: 72°C for 5:00 min.<br/ >
- Final elongation: 72°C for 5:00 min.<br/ >
- Final hold: 4°C for ∞.
<br/ >
<br/ >
* The controls were done as follows:
<br/ >
- Negative control: Blank<br/ >
- Positive control: Instead of using the rbs + 1 Fwd primer, it uses Prefix.<br/ >
<br/ >
* Tomorrow, I will prove by electrophoresis that the output is consistent :D.
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Revision as of 11:45, 17 September 2010

WiFi coli: A communicolight system <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

PCR day


  • Today, I wanted to do two PCRs: one in order to change the RBS of the luciferase as described in the parts registry, and another to conduct a punctual mutation for the emission spectrum of luciferase.


  • The first assay failed, as I missed the hot start step. The reaction was done as follows for 6.5 reactions using as a forward primer the rbs + 1 primer (syntethized to avoid the complications described in the parts registry above), and a dilution of DNA (5μL / 20 of H2O):


  • The Fwd primer sequence is: 5' -> 3' GCT CTA GAG AGG AAA CAG CTA TGG AAG ACG CCA AAA AC


--> Mix 1 for 30 μL <--

-H2O ------------> 74.75μl
-Buffer 3.3 -----> 39μl
-Mg(OAc)2 -----> 19.5μl
-dNTP's ---------> 16.5μl
-Forward -------> 16.5μl
-Reverse -------> 16.5μl
-DNA -----------> (2μl for the tubes that required it)


--> Mix 2 for 20 μL <-- (this reaction will be added after the hot start)

-H2O ---------------------> 68.25μl
-Buffer 3.3 --------------> 58.5μl
-rtTh polymerase -----> 3.25μl


  • The 30 cycles were programed as follows:



- Initialization step: 95°C for 5 min. (only the 1st mix)

- Hot start: Stop to add the second mix

- Denaturation step: 95°C for 45 seg.

- Annealing step: 60°C for 45 seg.

- Extension/elongation step: 72°C for 1:50 min.

- Final elongation: 72°C for 5:00 min.

- Final hold: 4°C for ∞.

  • The controls were done as follows:


- Negative control: Blank
- Positive control: Instead of using the rbs + 1 Fwd primer, it uses Prefix.

  • Tomorrow, I will prove by electrophoresis that the output is consistent :D.


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