User:Hana Benzeer/Notebook/SGM Summer Project/2011/06/15

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DIGESTION BBa_E0840 BBa_R0082 BBa_M30109 For the first composite between the E0840 and R0082 parts, as R0082 was chosen as the backbone and E0840 the downstream reporter. R0082 was digested with P and S To 30ul of R0082 we added 0.5ul, 100xBSA 5ul, buffer 2 1μL, PstI 1μL, SpeI 12.5μL, H2O The 50μL reaction was placed at 37C for 2 hrs E0840 was digested with P and X To 30ul of E0840 we added 0.5ul, 100xBSA 5ul, buffer 3 1μL, PstI 1μL, XbaI 12.5μL, H2O The 50μL reaction was placed at 37C for 2 hrs. Gel preperation prepared 1% and 2% agarose gel 1% gel, loading 1Kb ladder 50ul, R0082, P/S 5μL, R0082, P/S Blank 5μL, E0840, P/X Blank Blank Blank 2% gel, loading 100 bp ladder 50ul, E0840, P/X 5μL, E0840, P/X Blank Blank Blank Blank Blank Gel purification the bands were cut with scapel from the gels. E0840, 900bp R0082, ~3kb the method, book Ligation To ligate the cognate promnoter R0082 with reporter,E0840. Add 1μL of R0082 backbone Add 7μL of E0840 insert Add 1μL of ligase buffer Add 1μL of ligase Incubate at room temp for 2 hrs Transformation To 50μL of competent cell (XL-1 Blue) Add 10μL of ligase reaction Place for 5 min on ice Place in the 42°C waterbath for 1 min Put back on ice for 5 min Add 250μL Soc Place in the shaker for 1 hr Plate on agar with appropriate antibiotic (Ampicillin) using glass beads Put in the incubater at 37°C