User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/08/23

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I just wanna ligate LRE, that will be the best experiment for me...


  • The first thing, I ran a gel to quantify my vectors and inserts as is observed here:




  • The lanes were as follows:


1. Ladder

2. J23100

3. pSB1C3

4. LRE restricted with XBA I and PST I

5. LRE restricted with ECO RI and PST I

6-14. Claudia's and Augusto's samples


Looking for ligation...


  • The next reactions describe the ligations to be done for a total of 20μl.


    • 1: LRE (XBA I/PST I) with J23100 labeled as LRE + J23100 Mar and date.


-H2O -----------------------> 7μL
-Buffer ligase -----------> 4μL
-Vector (plasmid) -----> 3μL
-Insert (LRE) ---> 5μL
-Ligase --------------------> 1μL

    • 2: LRE (ECO RI/PST I) with plasmid 17 labeled as LRE + p17 Mar and date.


-H2O -----------------------> 7μL
-Buffer ligase -----------> 4μL
-Vector (plasmid) -----> 3μL
-Insert (LRE) ---> 5μL
-Ligase --------------------> 1μL

    • 3: Control (J23100) labeled as Co J23100 Mar and date.


-H2O -----------------------> 13μL
-Buffer ligase -----------> 4μL
-Vector (plasmid) -----> 2μL
-Ligase --------------------> 1μL

    • 4: Control (plasmid 17) labeled as Co p17 Mar and date.


-H2O -----------------------> 13μL
-Buffer ligase -----------> 4μL
-Vector (plasmid) -----> 2μL
-Ligase --------------------> 1μL

  • The reactions were left at 16°C ON.



Preparing the assay


  • Tomorrow, finally, Dr. Wood will help me to test if the luciferase has mutated. The assay we are planning to use is the same applied by the Edinburgh team. As it indicates ON incubation of the colonies to test, I inoculated 5ml of LB with 4μl of ampicilin (10 strains for mutated luciferase and 5 for wild type).


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