User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/06/17

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Starting with the punctual mutation on luciferase


  • As referred | previously, the mutation on luciferase requires the amplification by PCR of the two fragments so that, when we use only the prefix and suffix, the mutation is there. I did the following reactions for the PCR using both Pfx and rtTh as a control.


  • 1. Pfx with Prefix and the primer for the mutation (Reverse) with DNA dilution of 1/5.


  • 2. Pfx with Prefix and the primer for the mutation (Reverse).


  • 5. Pfx with primer for the mutation (Forward) and Suffix with DNA dilution of 1/5.


  • 6. Pfx with primer for the mutation (Forward) and Suffix.


-H2O ----------------------------------------> 38.2μl
-Buffer Pfx ---------------------------------> 5μl
-MgSO4 ------------------------------------> 1μl
-dNTP's (10μM)---------------------------> 1.5μl
-Primer mix (10μM) ----------------------> 1.5μl
-DNA (PCR ligation product) -----------> 2μl
-Pfx -----------------------------------------> 0.8μl

  • 3. Pfx Negative control using both, Prefix and primer for the mutation (Reverse) and primer for the mutation (Forward) and Suffix.


-H2O ----------------------------------------> 38.2μl
-Buffer Pfx ---------------------------------> 5μl
-MgSO4 ------------------------------------> 1μl
-dNTP's (10μM)---------------------------> 1.5μl
-Primer mix 1 (10μM) --------------------> 1.5μl
-Primer mix 2 (10μM) --------------------> 1.5μl
-DNA (PCR ligation product) -----------> NONE
-Pfx -----------------------------------------> 0.8μl

  • 4. Pfx Positive Control using Prefix and Suffix.


-H2O ----------------------------------------> 38.2μl
-Buffer Pfx ---------------------------------> 5μl
-MgSO4 ------------------------------------> 1μl
-dNTP's (10μM)---------------------------> 1.5μl
-Primer mix (10μM) ----------------------> 1.5μl
-DNA (plasmid 6) -------------------------> 2μl
-Pfx -----------------------------------------> 0.8μl

  • 7. rtTh Negative Control using both, Prefix and primer for the mutation (Reverse) and primer for the mutation (Forward) and Suffix.


--> Mix 1 for 30 μL <--

-H2O ------------> 11.5μl
-Buffer 3.3 -----> 6μl
-Mg(OAc)2 -----> 3μl
-dNTP's ----------> 2.5μl
-Primer mix 1 --> 2.5μl
-Primer mix 2 --> 2.5μl
-DNA ------------> NONE


--> 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


  • 8. rtTh Positive Control using Prefix and Suffix as primers and plasmid 6.


--> Mix 1 for 30 μL <--

-H2O ------------> 13μl
-Buffer 3.3 -----> 6μl
-Mg(OAc)2 -----> 3μl
-dNTP's ----------> 2.5μl
-Primer mix 1 --> 2.5μl
-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


  • 9. rtTh with Prefix and the primer for the mutation (Reverse) with DNA dilution of 1/5.


  • 10. rtTh with Prefix and the primer for the mutation (Reverse).


  • 11. rtTh with primer for the mutation (Forward) and Suffix with DNA dilution of 1/5.


  • 12. rtTh with primer for the mutation (Forward) and Suffix.


--> Mix 1 for 30 μL <--

-H2O ------------> 13μl
-Buffer 3.3 -----> 6μl
-Mg(OAc)2 -----> 3μl
-dNTP's ----------> 2.5μl
-Primer mix ----> 2.5μl
-DNA ----> 2μl of a 1/5 dilution


--> 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 35 cycles were programed as follows:



- Initialization step: 94°C for 4 min. (only the 1st mix)

- Hot start: Stop to add the second mix

- Denaturation step: 94°C for 15 seg.

- Annealing step: 60°C for 30 seg.

- Extension/elongation step: 68°C for 1 min.

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

- Final hold: 4°C for ∞.