Team:Calgary/16 June 2009
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- | + | Construction Digest to Move From pCR2.1-TOPO to psB1AC3 Vector | |
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- | + | *Objective: The LuxOD47E gene in now in linear form with the Biopbrick restriction sites attatched. We now want to get it into the psB1AC3 vector, | |
+ | *Protcol: To do this, we will digest both the gene and the vector in two ways: one way with EcoRI and PstI and one way with XbaI and PstI | ||
+ | *Insert tube 1- DNA to approximately [600ng/uL]= 3.1uL | ||
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+ | ddH2O up to 35 uL- 31.9 uL | ||
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+ | 4 uL REact 2 Buffer | ||
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+ | 1 uL XbaI restriction enzyme | ||
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+ | 1 uL PstI restriction enzyme | ||
+ | *Insert tube 2- same as for tube one, but with EcoRI restriction enzyme in place of XbaI restriction enzyme. | ||
+ | *Vector tube 1- psB1AC3 to [200ng/uL], REact 2 Buffer, XbaI restriction enzyme and PstI restriction enzyme | ||
+ | *Vector tube 2- same as for tube one, but with EcoRI restriction enzyme in place of XbaI restriction enzyme. | ||
+ | *Placed in 37 C waterbath for 2,5 hours followed by heat deactivation for 10 minutes in a 65 C heating block. | ||
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Revision as of 05:31, 21 August 2009
UNIVERSITY OF CALGARY