User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/14

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PCR day

  • Today, I wanted to do two PCRs: one in order to change the RBS of the luciferase as described in the parts registry, and another to conduct a punctual mutation for the emission spectrum of luciferase.

  • The first assay failed, as I missed the hot start step. The reaction was done as follows for 6.5 reactions using as a forward primer the rbs + 1 primer (syntethized to avoid the complications described in the parts registry above), and a dilution of DNA (5μL / 20 of H2O):

  • The Fwd primer sequence is: 5' -> 3' GCT CTA GAG AGG AAA CAG CTA TGG AAG ACG CCA AAA AC

--> Mix 1 for 30 μL <--

-H2O ------------> 74.75μl
-Buffer 3.3 -----> 39μl
-Mg(OAc)2 -----> 19.5μl
-dNTP's ---------> 16.5μl
-Forward -------> 16.5μl
-Reverse -------> 16.5μl
-DNA -----------> (2μl for the tubes that required it)

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

-H2O ---------------------> 68.25μl
-Buffer 3.3 --------------> 58.5μl
-rtTh polymerase -----> 3.25μl

  • The 30 cycles were programed as follows:

- Initialization step: 95°C for 5 min. (only the 1st mix)

- Hot start: Stop to add the second mix

- Denaturation step: 95°C for 45 seg.

- Annealing step: 60°C for 45 seg.

- Extension/elongation step: 72°C for 1:50 min.

- Final elongation: 72°C for 5:00 min.

- Final hold: 4°C for ∞.

  • The controls were done as follows:

- Negative control: Blank
- Positive control: Instead of using the rbs + 1 Fwd primer, it uses Prefix.

  • Tomorrow, I will prove by electrophoresis that the output is consistent :D.