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

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And now, click beetle time!!!
  --> 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 <br/ > -Buffer 3.3 -> 9μl <br/ > -rTth > 0.5μl <br/ > <br/ > <br/ > <br/ > - Initialization step: 94°C for 4 min. (only the 1st mix)<br/ >
 * As the primers to amplify the click beetle luciferase are already here, I did a PCR with rTth:
 * The 30 cycles were programmed as follows:

- Hot start: Stop to add the second mix<br/ >

- Denaturation step: 94°C for 45 seg. <br/ >

- Annealing step: 55-60°C for 45 seg. <br/ >

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

- Final elongation: 72°C for 10:00 min.<br/ >

- Final hold: 4°C for ∞. <br/ > <br/ > <br/ > <br/ > <br/ > <br/ > 1. Ladder.<br/ >
 * 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):<br/ >
 * Where the lanes are as follows:<br/ >

2. Negative control.<br/ >

3. Green luciferase with anneling temperature at 55°C.<br/ >

4. Red luciferase with anneling temperature at 55°C.<br/ >

5. Green luciferase with anneling temperature at 60°C.<br/ >

6. Red luciferase with anneling temperature at 60°C.<br/ >

7. Positive control: mutated (red) luciferase of Photinus pyralis. <br/ > <br/ > <br/ > <br/ > <br/ >
 * 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.<br/ >

L.R.E.
<br/ > <br/ > <br/ > <br/ > 1. Ladder.<br/ >
 * 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:<br/ >
 * The lanes were as follows:<br/ >

2, 3, 4. LRE restricted with ECO RI and PST I in pSB1C3.<br/ >

5, 6, 7. LRE restricted with XBAI and PST I in BBa_J61002 with J23100 promoter. <br/ > <br/ > <br/ > <br/ >
 * 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. <br/ >


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