User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/09/07

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And now, click beetle time!!!


  • As the primers to amplify the click beetle luciferase are already here, I did a PCR with rTth:


--> Mix 1 for 30 μL <--

-H2O ----------------> 9μl
-Buffer 3.3x --------> 6μl
-Mg(OAc)2 ----------> 3μl
-dNTP's -------------> 5μl
-CBluc Fw ----------> 3μl
-CBluc Rv -----------> 3μl
-DNA (1/50)---------> 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: 55-60°C for 45 seg.

- Extension/elongation step: 72°C for 1:50 min.

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

- Final hold: 4°C for ∞.

  • The following gel shows the PCR product which was obtained according to the expected size (1629 for click beetle luciferase and ~1800 for firefly + double terminator luciferase):


CBlucPCR.JPG

  • Where the lanes are as follows:


1. Ladder.

2. Negative control.

3. Green luciferase with anneling temperature at 55°C.

4. Red luciferase with anneling temperature at 55°C.

5. Green luciferase with anneling temperature at 60°C.

6. Red luciferase with anneling temperature at 60°C.

7. Positive control: mutated (red) luciferase of Photinus pyralis.


  • The product was purified using the High PCR purification kit of Roche and product was labeled and stored as Green/Red CB or Mut Luz PCR purif Mar and date.



L.R.E.


  • I started the day by doing a restriction of LRE with both pSB1C3 and with BBa_J61002, so that I can check that the transformation went out well. Restrictions were done for 20μl and incubated for 4hrs at 37°C. Afterwards, a gel was run:


LREPlasmPurif.JPG

  • The lanes were as follows:


1. Ladder.

2, 3, 4. LRE restricted with ECO RI and PST I in pSB1C3.

5, 6, 7. LRE restricted with XBAI and PST I in BBa_J61002 with J23100 promoter.

  • As observed, only strain 10 came out as expected. Following this, strains 3,5, and 7 of LRE with pSB1C3 were inoculated in 4mL of LB with 4μL of Chloramphenicol.