User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/08/02

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...and the work keeps increasing with luciferases
  --> Mix 1 for 30 μL <--   -H2O > 9μl  -Buffer 3.3x > 6μl  -Mg(OAc)2 --> 3μl -dNTP's -> 5μl  -Primer Fw --> 3μl  -Suffix ---> 3μl  -DNA (3/50)-> 1μl   --> Mix 2 for 20 μL <-- (this reaction will be added after the hot start)   -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/ >
 * As my mutated luciferase stock was almost over, and things still keep interfering with my constructions, I had to amplify more product. The reaction was done as follows, where one tube (1) used the first part of mutated luciferase as forward primer, another a dilution of 1/2 of it (2), and the positive control is normal luciferase using a DNA dilution of 1/10:
 * The 30 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/ > <br/ > <br/ > <br/ > <br/ > 1. Ladder.<br/ >
 * After the PCR reaction was finished, I ran a gel to verify that everything went out well:<br/ >
 * The lanes were as follows:<br/ >

2. Mutated luciferase (1).<br/ >

3. Mutated luciferase (2). <br/ >

4. Negative control. <br/ >

5. Positive control. <br/ > <br/ > <br/ >


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