User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/05/31

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Ready for ligation!!!

  • What I did today was a restriction of the PCR purified product (luciferase) with ECO RI and SPEI for a total of 40μl and stored as ECO RI y SPE I Prod. luc. PCR Mar and date (1-4). The reaction is described next:

-H2O ---------> 18μl
-BSA ---------> 1μl
-Buffer ------> 4μl
-ECO RI -----> 1μl
-SPEI --------> 1μl
-DNA --------> 15μl

  • In order to quantify the vector-insert relation for the ligation, I ran a 2% agarose gel for 50 min at 90V:

TT luc.JPG

  • The lanes are as follow:

1. Ladder
2. Blank
3. Double terminator BBa_B0015
4. Restriction of luciferase
5. Negative control (water)
6. Positive control (plasmid with luciferase but without restriction)

  • Finally, I did the ligation as follows for a total of 20μL:

-H2O ----------------------> 12μL
-Buffer ligase ----------> 2μL
-Vector (plasmid) ----> 2μL
-Insert (luciferase) --> 3μL
-Ligase -------------------> 1μL

  • The mix was incubated at 22°C for 10 min and at 70°C for 10 min to inactivate the enzyme.