User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/24

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  • Today, I ran a gel to check if my PCR product was there:

Mut luz.JPG

  • The lanes were as follows:

1. Ladder.
2. Luciferase with the mutation (complete).
3. Positive control.

Another PCR

  • After the success, I went to see Miguel to check the next steps. This involves doing a PCR with a 5/100 DNA dilution (of the previous PCR product) using rtTh. The reaction is defined as follows and I did two reactions with the dilution and the respective controls.

--> Mix 1 for 30 μL <--

-H2O ------------> 9μl
-Buffer 3.3 -----> 6μl
-Mg(OAc)2 -----> 3μl
-dNTP's ----------> 4μl
-Prefix --------> 3μl
-Suffix --------> 3μl
-DNA ------------> 2μl

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

-H2O ---------------------> 10.5μl
-Buffer 3.3 --------------> 9μl
-rtTh polymerase ------> 0.5μl

  • The gel (ran for 50min at 90V) shows that the two expected products are there:

Mut luz diluida.JPG

  • The lanes are:

1. Ladder.
2-7. Jorge's experiment.
8. Negative control.
9. Positive control.
10 and 11. Mutated luciferase PCR product.