User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/09/01

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I'm kinda busy...


  • Today, I ran a gel to check that my PCR product was there.


PCRmasluc.JPG

  • The lanes were ladder, mutated luc., negative control, mutated luc. (2), and normal luciferase (3).


  • As there is a lot of non-specific product, I will repeat the PCR but using the RBS instead of the preffix, and the RV kanamycin cassette primer instead of the suffix.



--> Mix 1 for 30 μL <--

-H2O ----------------> 9μl
-Buffer 3.3x --------> 6μl
-Mg(OAc)2 ----------> 3μl
-dNTP's -------------> 5μl
-Primer Fw ----------> 3μl
-Primer Rv ----------> 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 35 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 ∞.

Transforming... again


  • Today, Melvin retransformed LRE as described previously.