User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/11

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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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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Starting with the PCRs
    --> Mix for 30 μL <--   -H2O > 40.5μl  -Buffer 3.3 -> 18μl  -MgCl2 --> 9μl  -dNTP's -> 6μl  -Forward ---> 6μl  -Reverse ---> 6μl  -DNA > (1μl for the tubes that require it)  -Taq pol > 1.5μl  <br/ > <br/ > The 30 cycles were programed as follows: <br/ > <br/ > - Initialization step: 95°C for 4:30 min. <br/ > - Denaturation step: 95°C for 45 seg. <br/ > - Annealing step: 60°C for 45 seg. <br/ > - Extension/elongation step: 72°C for 2:10 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/ > <br/ > - Negative control: Blank<br/ > - Positive control: Plasmid 18 (construction of kanamycin).<br/ > <br/ > <br/ > <br/ > <br/ > 1. Negative control<br/ > 2. Ladder<br/ > 3. and 5. PCR luciferase and TT product<br/ > 4. Positive control<br/ > <br/ >
 * Today, I did a PCR of the restriction done on luciferase with ECO RI and PST I.
 * The reaction was done as follows for 3 reactions using as a forward primer the prefix and as a reverse primer which consists of a kanamycin with a terminator and only anneals and amplifies DNA if there is a double terminator, this would indicate that ligation was done successfully:
 * The primer sequence is: 5' -> 3' : CTG CAG CGG CCG CTA CTA GTA TAT AAA CGC AGA AAG GCC CAC CC
 * To check that the PCR product was there, I ran an 8% agarose gel at 90V for 50min.
 * The lanes are:


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