User:Mariana Ruiz Velasco L./Notebook/IGEM 2010/Wet lab journal/2010/08/06

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More and more and more...
  1. Mutated luciferase with ECO RI and PST I stored as mut-luz R-> ECO y PST Mar and the date.  -H2O > 12μl -Buffer 3 --> 3μl -BSA ---> 1μl -DNA ---> 10μl -ECO I -> 2μl -PST I --> 2μl  2. pSB1C3 with ECO RI and PST I stored as p17 R-> ECO y PST Mar and the date.   -H2O > 12μl -Buffer 3 --> 3μl<br/ > -BSA > 1μl<br/ > -DNA > 10μl<br/ > -ECO RI > 2μl<br/ > -PST I ---> 2μl <br/ > <br/ > <br/ > <br/ > <br/ > <br/ > <br/ > 1,7. Ladder. <br/ >
 * I'm ready to do the restrictions and ligations (FINALLY!!!) for the pBSKII (+/-) vector so that I can finally prove the mutation. At the same time, I will be using pSB1C3, as it is the standard for iGEM's 2010 DNA submission. The restrictions:
 * The gel:
 * The lanes:

2. Mutated luciferase restricted with ECO RI and PST I.<br/ >

3. p17 restricted with ECO RI and PST I. <br/ >

4,5,6. Things from Zepeda and Augusto. <br/ > <br/ > <br/ >

Ligations... again :D
<br/ > <br/ > -H2O ---> 9μL<br/ > -Buffer ligase ---> 2μL<br/ > -Vector (plasmid) -> 2μL<br/ > -Insert (luciferase) ---> 6μL<br/ > -Ligase > 1μL<br/ > <br/ > <br/ > -H2O ---> 8μL<br/ > -Buffer ligase ---> 2μL<br/ > -Vector (plasmid) -> 3μL<br/ > -Insert (luciferase) ---> 6μL<br/ > -Ligase > 1μL<br/ > <br/ > <br/ > -H2O ---> 10μL<br/ > -Buffer ligase ---> 2μL<br/ > -Vector (plasmid) -> 2μL<br/ > -Insert (luciferase) ---> 5μL<br/ > -Ligase > 1μL<br/ > <br/ > <br/ > -H2O ---> 9μL<br/ > -Buffer ligase ---> 2μL<br/ > -Vector (plasmid) -> 3μL<br/ > -Insert (luciferase) ---> 5μL<br/ > -Ligase > 1μL<br/ > <br/ > <br/ > -H2O ---> 15μL<br/ > -Buffer ligase ---> 2μL<br/ > -Vector (plasmid) -> 2μL<br/ > -Ligase > 1μL<br/ > <br/ > <br/ > -H2O ---> 14μL<br/ > -Buffer ligase ---> 2μL<br/ > -Vector (plasmid) -> 3μL<br/ > -Ligase > 1μL<br/ > <br/ > <br/ >
 * 1: luciferase (green) with pBSKII
 * 2: luciferase (green) with plasmid 17
 * 3: luciferase (red) with pBSKII
 * 4: luciferase (red) with plasmid 17
 * 5: Control (pBSKII)
 * 6: Control (plasmid 17)
 * The reactions were left at 4°C during the whole weekend.


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