User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/04/15

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First restriction :D

  • I realized a double restriction using ECO RI-HF and PST I for a total of 20μl as follows:

-H2O -------> 10μl
-Buffer 2 --> 2μl
-DNA -------> 5μl
-ECO RI ---> 1μl
-PSTI ------> 1μl

  • Restrictions were incubated at 37°C ON(overnight).

Defosfating restrictions

  • Continuing with the experiment, I defosfated restrictions of plasmids 17, 18 (jorge), 18 and 30. The reaction was as follows for a total of 30μL.

-H20 -------------> 11μL
-Buffer ----------> 3μL
-DNA ------------> 15μL
-Defosfatase --> 1μL

  • The reaction was incubated at 37°C for 15 min. and at 65°C for 5 min to inactivate the enzyme.