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Minnesota/22 July 2008
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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. | |'''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. | ||
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| - | |'''5. Vector Dephosphorylation:''' Same dephos. procedure used on GFP:Terminator | + | |'''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: | |'''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: | ||
Revision as of 21:12, 22 July 2008
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| 1. Pick Colonies from plates made 07-21-2008 Start cultures. |
| 2. Plasmid prep: Prep RFP, YFP, GFP, TetR promoter, Terminator. Follow QIA miniprep procedure --> 1hr long. |
| 3. Spec the prep products: Using spectrophotometry, the DNA concentration of the plasmid prep products were measured. |
| 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. |
| 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. |
| 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: |
| a. BV + TetR:p22 promoter + RFP |
| b. BV + TetR:p22 promoter + YFP |
| c. LacI:LambdacI + GFP:Terminator |
| d. LacI Promoter + p22 cII + BV |
| 7. Transformation: Procedure performed in 30 minutes. Transform all ligation products. Incubate in 2mL LB cultures for 2 hours @37C with shaking @ 220rpm's. |
| 8. Plate transformations: Plate transformation cultures. |
| 9. Prepare sequencing reactions: Prepare sequencing rxns IF POSSIBLE. |
