User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/07/02

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Working, working, working on E. coli
 
 * As the work that I have been doing the previous days hasn't turned out as expected, we decided to double efforts so that, before next week ends, we have tested the red luciferase. This double effort consists on repeating the experiments since the restriction of pSB1T3 a.k.a. plasmid 18 and the one with the constitutive promoter, and to do a PCR of the plasmids extracted from the already transformed strains. 

 1. Plasmid with constitutive promoter stored as J23106 Mar SPE y PST and the date.  -H2O ---> 8μl
 * I started the day by making a restriction for a total of 30μl as follows:

-Buffer 2 --> 3μl

-BSA --> 1μl

-DNA ---> 15μl

-SPE I -> 1.5μl

-PST I --> 1.5μl  2. Plasmid 18 stored as P 18 Mariana XBA y PST and the date.<br/ > <br/ > -H2O ---> 8μl<br/ >

-Buffer 2 --> 3μl<br/ >

-BSA --> 1μl<br/ >

-DNA ---> 15μl<br/ >

-XBA I -> 1.5μl<br/ >

-PST I --> 1.5μl<br/ > <br/ > <br/ >
 * Restrictions were incubated at 37°C for 4 hours and inactivated for 10 min at 80°C.<br/ >

<br/ >
 * I repeated the extraction of plasmid from plate 1 (luciferase in plasmid with J23106), as I went wrong in a step. This time I only used 1.2, 1.7, and 1.9.

PCR!!!
<br/ > <br/ >
 * I also did a PCR reaction as follows:

--> Mix 1 for 50 μL <-- <br/ > <br/ > -H2O -> 33μl <br/ > -Buffer ---> 5μl <br/ > -MgCl2 ---> 2.5μl <br/ > -dNTP's --> 2.5μl <br/ > -Prefix > 2.5μl <br/ > -Suffix > 2.5μl <br/ > -DNA --> 1μl <br/ > -Taq --> 1μl<br/ > <br/ >

<br/ >
 * As a control to check that luciferase and the double terminator were there, I used also rbs+1 forward primer and the kanamycin primer (only in plasmid 18).

<br/ > - Initializing step: 94°C for 5 min.<br/ >
 * We program the thermo-cycler as follows for 35 cycles:

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

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

- Extension/elongation step: 72°C for 2:00 min.<br/ >

- Final elongation: 72°C for 5:00 min.<br/ >

- Final hold: 4°C for ∞. <br/ > <br/ >

Preparing the ligation
<br/ > <br/ > - H2O > 11μl<br/ >
 * The first thing to do was to dephosphate the plasmids to prevent them from re-ligating themselves. The reaction for 30μL was done as follows:<br/ >

- Buffer phosphatase > 3μl<br/ >

- DNA ---> 15μl<br/ >

- Phosphatase > 1μl<br/ > <br/ > <br/ > <br/ > <br/ >
 * The tubes (labeled as the plasmid defosfat. Mariana and the date) were incubated at 37°C for 20 min and the enzyme was inactivated at 65°C for 10 min. <br/ >
 * I finally ran a gel at 80V for 1hr which didn't run well.


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