| 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. Spec the prep products: Using spectrophotometry, the DNA concentration of the plasmid prep products were measured.
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4. 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. Follow the table below for double digest guidelines:
| Parts | 10x Buffer | BSA | H20 | DNA | RE 1 | RE 2
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| R2 | 5.0uL | 0.5uL | 40.5uL | 2.0uL | 1.0uL, Xba1 | 1.0uL, Pst1
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| Y+2 | 5.0uL | 0.5uL | 40.5uL | 2.0uL | 1.0uL, Xba1 | 1.0uL, Pst1
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| Y+4 | 5.0uL | 0.5uL | 39.5uL | 3.0uL | 1.0uL, Xba1 | 1.0uL, Pst1
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| R+4 | 5.0uL | 0.5uL | 9.5uL | 33.0uL | 1.0uL, Xba1 | 1.0uL, Pst1
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| Lac Pro. | 5.0uL | 0.5uL | 41.5uL | 1.0uL | 1.0uL, EcoRI | 1.0uL, Spe1
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| p22 cII | 5.0uL | 0.5uL | 39.5uL | 3.0uL | 1.0uL, Xba1 | 1.0uL, Pst1
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| BV | 5.0uL | 0.5uL | 9.5uL | 33.0uL | 1.0uL, EcoRI | 1.0uL, Pst1
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| 5. Vector Dephosphorylation: Same dephos. procedure used on GFP:Terminator sample and BV sample. After dephosphorylation, incubate @37C for 30 mins. Heat inactivate dephosphorylation enzyme for 15 mins in 65C water bath.
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| 6. 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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| 7. 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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| 8. Plate transformations: Plate transformation cultures.
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| 9. Prepare sequencing reactions: Prepare sequencing rxns IF POSSIBLE.
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