User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/08/05

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More product please...
  --> 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 5 min. (only the 1st mix)<br/ >
 * As my final concentrations were so low, I decided to repeat the PCR of the other day, but now putting 4 reactions of the mutated luciferase and keeping the same controls.
 * 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 3 min.<br/ >

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

- Final hold: 4°C for ∞. <br/ > <br/ > <br/ > <br/ > <br/ > <br/ >
 * The gel shows that everything went ok :D<br/ >
 * The pcr products were purified with Roche's® kit and stored as Mut-luz Prod PCR purif Mar and date under the number 2-O and 2-P


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