User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/07/08

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 * style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]] WiFi coli: A communi-colight system
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More work...
     --> Mix 1 for 30 μL <--   -H2O > 8μl  -Buffer 3.3 -> 6μl  -Mg(OAc)2 -> 3μl  -dNTP's --> 5μl  -Primer 1 --> 3μl  -Primer 2 --> 3μl  -DNA > 2μl  <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 polymerase --> 0.5μl <br/ > <br/ > <br/ > <br/ > <br/ > - Initialization step: 94°C for 4:30 min. <br/ >
 * I started the day by plating the transformed strains that did grow well. Still, results weren't as expected :(
 * 1. rtTh Negative Control using Prefix and Suffix.
 * 2. rtTh Positive Control using Prefix and Suffix as primers and plasmid 18 with 7+6+5.
 * 3. rtTh with Kanamicin primer and the primer for the mutation of the rbs with DNA dilution of 1/10.<br/ >
 * The 30 cycles were programed as follows:

- Hot start: Stop to add the second mix 2. <br/ >

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

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

- Extension/elongation step: 72°C for 3:50 min.<br/ >

- Final elongation: 72°C for 10:00 min.<br/ >

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


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