User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/23

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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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Another day, another gel
   
 * As the PCR went out well, today I asked Miguel about our next steps. First, I purified the PCR product and gather it for a total of 60μl per piece. Second, I ran a gel (last two lanes) to check the concentration of my purified product:

Planning the punctual mutation: The protocol
  --> Mix 1 for 45 μL <--   -PCR 1 --> 5μl -PCR 2 --> 5μl  -H2O > 25μl  -Buffer 10x > 5μl  -MgCl2 (50mM) ---> 2.5μl -dNTP's (0.4mM) --> 2.5μl   Note: the positive control has water instead of PCR 2 product. <br/ > <br/ > - 95°C for 5 min. <br/ >
 * The first thing to do is to make a mix as follows:
 * Second, we program the thermo-cycler as follows for 1 cycle:

- 60°C for 30 sec. <br/ >

- 95°C for 5 min. <br/ >

- 60°C for 30 sec. <br/ >

- 4°C for 2:00 min. <br/ > <br/ > <br/ > - Denaturation step: 95°C for 30 sec. (In this cycle we add 4μl of primer mix with prefix and suffix) <br/ >
 * The third step was to add 1μl of Taq after the cycle was at 68°C for 2:30 min and was about to start 30 cycles as follows:

- Annealing step: 60°C for 30 sec. <br/ >

- Extension/elongation step: 72°C for 1:30 min.<br/ >

- Final elongation: 72°C for 5:00 min.<br/ >

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


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