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

WiFi coli: A communi-colight system

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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): 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: 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.