User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/08/02

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...and the work keeps increasing with luciferases

  • As my mutated luciferase stock was almost over, and things still keep interfering with my constructions, I had to amplify more product. The reaction was done as follows, where one tube (1) used the first part of mutated luciferase as forward primer, another a dilution of 1/2 of it (2), and the positive control is normal luciferase using a DNA dilution of 1/10:

--> 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
-Buffer 3.3 -----> 9μl
-rTth ------------> 0.5μl

  • The 30 cycles were programmed as follows:

- Initialization step: 94°C for 4 min. (only the 1st mix)

- Hot start: Stop to add the second mix

- Denaturation step: 94°C for 45 seg.

- Annealing step: 60°C for 45 seg.

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

- Final elongation: 72°C for 7:00 min.

- Final hold: 4°C for ∞.

  • After the PCR reaction was finished, I ran a gel to verify that everything went out well:

  • The lanes were as follows:

1. Ladder.

2. Mutated luciferase (1).

3. Mutated luciferase (2).

4. Negative control.

5. Positive control.

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