User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/11

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Starting with the PCRs

  • Today, I did a PCR of the restriction done on luciferase with ECO RI and PST I.

  • The reaction was done as follows for 3 reactions using as a forward primer the prefix and as a reverse primer which consists of a kanamycin with a terminator and only anneals and amplifies DNA if there is a double terminator, this would indicate that ligation was done successfully:

  • The primer sequence is: 5' -> 3' : CTG CAG CGG CCG CTA CTA GTA TAT AAA CGC AGA AAG GCC CAC CC

--> Mix for 30 μL <--

-H2O ------------> 40.5μl
-Buffer 3.3 -----> 18μl
-MgCl2 ----------> 9μl
-dNTP's ---------> 6μl
-Forward -------> 6μl
-Reverse -------> 6μl
-DNA ------------> (1μl for the tubes that require it)
-Taq pol --------> 1.5μl

The 30 cycles were programed as follows:

- Initialization step: 95°C for 4:30 min.
- Denaturation step: 95°C for 45 seg.
- Annealing step: 60°C for 45 seg.
- Extension/elongation step: 72°C for 2:10 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: Plasmid 18 (construction of kanamycin).

  • To check that the PCR product was there, I ran an 8% agarose gel at 90V for 50min.

PCR lucif TT 11.JPG

  • The lanes are:

1. Negative control
2. Ladder
3. and 5. PCR luciferase and TT product
4. Positive control