User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/09/01

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I'm kinda busy...
       --> Mix 1 for 30 μL <--   -H2O > 9μl  -Buffer 3.3x > 6μl  -Mg(OAc)2 --> 3μl -dNTP's -> 5μl  -Primer Fw --> 3μl  -Primer Rv --> 3μl  -DNA (3/50) -> 1μl<br/ > <br/ > <br/ > --> Mix 2 for 20 μL <-- (this reaction will be added after the hot start) <br/ > <br/ > -H2O > 10.5μl <br/ > -Buffer 3.3 -> 9μl <br/ > -rTth > 0.5μl <br/ > <br/ > <br/ > <br/ > - Initialization step: 94°C for 4 min. (only the 1st mix)<br/ >
 * Today, I ran a gel to check that my PCR product was there.<br/ >
 * The lanes were ladder, mutated luc., negative control, mutated luc. (2), and normal luciferase (3).
 * As there is a lot of non-specific product, I will repeat the PCR but using the RBS instead of the preffix, and the RV kanamycin cassette primer instead of the suffix.<br/ >
 * The 35 cycles were programmed as follows:

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

- Denaturation step: 94°C for 45 seg. <br/ >

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

- Extension/elongation step: 72°C for 2 min.<br/ >

- Final elongation: 72°C for 7:00 min.<br/ >

- Final hold: 4°C for ∞. <br/ > <br/ >

Transforming... again
<br/ > <br/ >
 * Today, Melvin retransformed LRE as described previously. <br/ >


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