User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/08/31

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I keep waiting here by the phone...

  • Today, Melvin and I noticed that something went out wrong with the transformations, so I decided we should run a gel to check both, restrictions of LRE to ligate and transform again, and my restrictions with the plasmids to check if I could ligate too. The gel showed that everything except for my luciferases were great.

Restrluc 31Ago10.JPG

  • Lanes:

1. Ladder.

2. J23100 with SpeI and PstI.

3. J23106 with SpeI and PstI.

4. Linearized pSB1C3 with Eco RI and Pst I.

5. Luciferase restricted with XbaI and PstI.

6. Mutated luciferase restricted with XbaI and PstI.

7. LRE restricted with XbaI and PstI.

8. LRE restricted with ECO RI and PstI.

  • As my mutated luciferase and wildtype stock were almost over, I had to amplify more product. The reaction was done as follows, where one tubes 1,2,3 contain the mutated luciferases, and tubes 4,5,+ the normal luciferase:

--> 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 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 ∞.