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

  • We program the thermo-cycler as follows for 30 cycles:


- Denaturation step: 94°C for 45 sec.

- Annealing step: 60°C for 45 sec.

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

- Final elongation: 72°C for 10:00 min.

- Final hold: 4°C for ∞.

¬¬ only one band was observed; however, it is from the right size.


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