User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/08/11

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Colony PCR reloaded
  
 * As my colonies did grow, it is time to check that the mutation on luciferase is done correctly by PCR means. The first thing was to extract the DNA by inoculating a colony of interest (5 for each plate) in 200μl of Tris-EDTA 10/1-NaCl 10mM, promote lysis leaving the cultures for 10min at 95°C, and centrifugate 2min at maximum speed.
 * The PCR reaction was done as follows:

--> Mix 1 for 50 μL <--   -H2O -> 24μl  -Buffer ---> 5μl  -MgCl2 ---> 2.5μl  -dNTP's --> 2.5μl  -Prefix > 2.5μl  -Suffix > 2.5μl  -DNA --> 10μl  -Taq --> 1μl  <br/ > - Denaturation step: 94°C for 45 sec.<br/ >
 * We program the thermo-cycler as follows for 30 cycles:

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

- Extension/elongation step: 72°C for 2:00 min.<br/ >

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

- Final hold: 4°C for ∞. <br/ > <br/ > ¬¬ only one band was observed; however, it is from the right size.


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