User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/18

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 * style="background-color: #EEE"|[[Image:Logo_light_minimal_full_res.jpg|150px]] WiFi coli: A communicolight system
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¬¬ Changing enzymes...
     1. Ladder.  2. Pfx with Prefix and the primer for the mutation (Reverse) (DNA dilution 1/5). 3. Pfx with Prefix and the primer for the mutation (Reverse). 4. Pfx Negative control. 5. Pfx Positive Control. 6. Pfx with primer for the mutation (Forward) and Suffix(DNA dilution 1/5). 7. Pfx with primer for the mutation (Forward) and Suffix. 8. rtTh Negative Control. 9. rtTh Positive Control. 10. rtTh with Prefix and the primer for the mutation (Reverse)(DNA dilution 1/5).<br/ > 11. rtTh with Prefix and the primer for the mutation (Reverse).<br/ > 12. rtTh with primer for the mutation (Forward) and Suffix(DNA dilution 1/5). <br/ > 13. rtTh with primer for the mutation (Forward) and Suffix.<br/ > 14. GFP of Arturo.<br/ > 15. Positive control of GFP (plasmid 6) of Arturo.<br/ > 16. Negative control of GFP of Arturo.<br/ > 17. LuxCDE of Miguel.<br/ > 18. LuxAB of Miguel.<br/ > 19. Ladder.<br/ > <br/ > <br/ >
 * Today, I again inoculated J23106 bacteria into 4ml of LB with 4μl of ampicilin and incubated at 37°C for 7hrs.
 * I also checked the PCR realized yesterday with the following gel of 8% agarose.
 * The lanes are:

PCR time
<br/ > <br/ >
 * As the gel shows, Pfx didn't amplify any of my reaction (though rtTh did). As the second enzyme cannot be used for a punctual mutation, I needed to repeat the PCR but now with Pfu-Turbo DNA Polymerase and with a dilution of 1/5 of the ligation with changed RBS PCR product.
 * 1. Using Prefix and the primer for the mutation (Reverse).
 * 2. Using primer for the mutation (Forward) and Suffix.
 * Positive control (+), uses prefix, suffix, and plasmid 6.
 * Negative control (-), uses 4 primers.

<br/ > --> Mix 1 for 30 μL <-- <br/ > <br/ > -H2O > 24.1μl <br/ > -Buffer Pfu-Turbo -> 2.5μl <br/ > -dNTP's (25mM each dNTP) -> 0.4μl <br/ > -DNA > 1μl <br/ > -Primer 1 (100ng/μl)--> 1μl <br/ > -Primer 2 (100ng/μl)--> 1μl <br/ > <br/ > <br/ > --> Mix 2 for 20 μL <-- (this reaction will be added after the hot start) <br/ > <br/ > -H2O -> 16.5μl <br/ > -Buffer Pfu --> 2.5μl <br/ > -Pfu-Turbo pol ---> 1μl <br/ > <br/ > <br/ > <br/ > - Initialization step: 95°C for 4 min. (only the 1st mix)<br/ >
 * The 30 cycles were programmed as follows:

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

- Denaturation step: 95°C for 30 seg. <br/ >

- Annealing step: 60°C for 30 seg. <br/ >

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

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

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


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