| 1. Pick Colonies from plates made 07-21-2008 Start cultures.
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| 2. Plasmid prep: Prep RFP, YFP, GFP, TetR promoter, Terminator. Follow QIA miniprep procedure --> 1hr long.
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| 3. Double digest: Follow Kat's DNA work procedure to perform Double digest on: LacI Promoter, p22 cII gene, RFP, YFP, BV (dual promoters, GFP:Term already d.dig.). Incubate digested products for 2 hours @37C. Heat inactivate digestion enzyme for 15 mins @ 65C water bath.
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| 4. Vector Dephosphorylation: Same dephos. procedure used on GFP:Terminator, BV. After dephosphorylation, incubate @37C for 30 mins. Heat inactivate dephosphorylation enzyme for 15 mins in 65C water bath.
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| 5. Ligation Reactions: Procedure performed in 20 minutes. Once all were ligated, were then incubated @ 16C for 1 hour. Heat inactivated enzyme @ 65C for 15 minutes. Ligated the following using L4 DNA Ligase:
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| a. BV + TetR:p22 promoter + RFP
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| b. BV + TetR:p22 promoter + YFP
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| c. LacI:LambdacI + GFP:Terminator
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| d. LacI Promoter + p22 cII + BV
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| 6. Transformation: Procedure performed in 30 minutes. Transform all ligation products. Incubate in 2mL LB cultures for 2 hours @37C with shaking @ 220rpm's.
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| 7. Plate transformations: Plate transformation cultures.
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| 8. Prepare sequencing reactions: Prepare sequencing rxns IF POSSIBLE.
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