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

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 * style="background-color: #EEE"|[[Image:Logo_light_minimal_full_res.jpg|150px]] WiFi coli: A communicolight system
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PCR day
    --> 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)   <br/ >
 * 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 2 for 20 μL <-- (this reaction will be added after the hot start) <br/ > <br/ > -H2O -> 68.25μl <br/ > -Buffer 3.3 --> 58.5μl <br/ > -rtTh polymerase -> 3.25μl <br/ > <br/ > <br/ > <br/ > <br/ > - Initialization step: 95°C for 5 min. (only the 1st mix)<br/ >
 * The 30 cycles were programed as follows:

- Hot start: Stop to add the second mix<br/ >

- Denaturation step: 95°C for 45 seg. <br/ >

- Annealing step: 60°C for 45 seg. <br/ >

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