User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/23

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Another day, another gel

  • As the PCR went out well, today I asked Miguel about our next steps. First, I purified the PCR product and gather it for a total of 60μl per piece. Second, I ran a gel (last two lanes) to check the concentration of my purified product:

PCR purif.JPG

Planning the punctual mutation: The protocol

  • The first thing to do is to make a mix as follows:

--> Mix 1 for 45 μL <--

-PCR 1 ----------------------------------> 5μl
-PCR 2 ----------------------------------> 5μl
-H2O ------------------------------------> 25μl
-Buffer 10x ----------------------------> 5μl
-MgCl2 (50mM) -----------------------> 2.5μl
-dNTP's (0.4mM) ----------------------> 2.5μl

Note: the positive control has water instead of PCR 2 product.

  • Second, we program the thermo-cycler as follows for 1 cycle:

- 95°C for 5 min.

- 60°C for 30 sec.

- 95°C for 5 min.

- 60°C for 30 sec.

- 4°C for 2:00 min.

  • The third step was to add 1μl of Taq after the cycle was at 68°C for 2:30 min and was about to start 30 cycles as follows:

- Denaturation step: 95°C for 30 sec. (In this cycle we add 4μl of primer mix with prefix and suffix)

- Annealing step: 60°C for 30 sec.

- Extension/elongation step: 72°C for 1:30 min.

- Final elongation: 72°C for 5:00 min.

- Final hold: 4°C for ∞.