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

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¬¬ Changing enzymes...


  • 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.


PCR Pfx.JPG

  • The lanes are:

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).
11. rtTh with Prefix and the primer for the mutation (Reverse).
12. rtTh with primer for the mutation (Forward) and Suffix(DNA dilution 1/5).
13. rtTh with primer for the mutation (Forward) and Suffix.
14. GFP of Arturo.
15. Positive control of GFP (plasmid 6) of Arturo.
16. Negative control of GFP of Arturo.
17. LuxCDE of Miguel.
18. LuxAB of Miguel.
19. Ladder.


PCR time


  • 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.


--> Mix 1 for 30 μL <--

-H2O ------------------------------------> 24.1μl
-Buffer Pfu-Turbo ---------------------> 2.5μl
-dNTP's (25mM each dNTP) ---------> 0.4μl
-DNA ------------------------------------> 1μl
-Primer 1 (100ng/μl)------------------> 1μl
-Primer 2 (100ng/μl)------------------> 1μl


--> Mix 2 for 20 μL <-- (this reaction will be added after the hot start)

-H2O ---------------------> 16.5μl
-Buffer Pfu --------------> 2.5μl
-Pfu-Turbo pol -----------> 1μl

  • The 30 cycles were programmed as follows:



- Initialization step: 95°C for 4 min. (only the 1st mix)

- Hot start: Stop to add the second mix

- Denaturation step: 95°C for 30 seg.

- Annealing step: 60°C for 30 seg.

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

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

- Final hold: 4°C for ∞.