Team:Calgary/9 July 2009
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- | + | *Today I ran a colony PCR of my J13002-LuxOD47E-B0015 colonies to verify the presence of the B0015 terminator in my construct. BBK-CP forward and reverse primers were used with p Taq and cycling conditions were as follows: 94°C for 6 min, 36x(94°C for 30 s, 55°C for 45s, 72°C for 90s), 72°Cfor 10 min, held at 4°C. PCR products were visualized on a 1% agarose gel run at 120 V with J13002-LuxOD47E and LuxOD47E as size controls. Gel is pictures below. Lanes 1-4 are trial one where the B0015 terminator was treated as the insert. Lanes 5-8 are trial 2 where the J13002-LuxOD47E construct was treated as the insert. Lane 9 is J13002-LuxOD47E and Lane 10 is LuxOD47E. Lane 11 is blank and Lane 12 is the negative control. | |
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+ | *Because all bands in the first 9 lanes appear to be the same size as the band in lane 9 with J13002-LuxOD47E, we can conclude that the B0015 terminator was not successfully cloned in and we will have to go back to contruction again. | ||
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+ | *Performed a restriction digest with EcoRI, XbaI and SpeI of B0015 and the J13002-LuxOD47E construct, digesting the insert with EcoRI and SpeI and digesting the recipient with EcoRI and XbaI. Left the digestioin in the 37C waterbath for 2 hours, transformed into TOP10 cells on both AC and C plates and left for overnight growth. | ||
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Revision as of 19:57, 20 August 2009
UNIVERSITY OF CALGARY