Woohoo!!!!
- Today, I ran a gel to check if my PCR product was there:
- 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:
1. Ladder.
2-7. Jorge's experiment.
8. Negative control.
9. Positive control.
10 and 11. Mutated luciferase PCR product.
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