| 1. Dephosphorylation: Continued with double digestion base vector product from 07-09-2008. Used Antartic phosphoylase to dephosphorylate the base vector. Want to dephosphorylate the base vector to prevent it from closing - want it to stay a linear digested product. After dephosphorylation, the base vector was then incubated @ 37C for 30 minutes. Sample was then placed in a heat bath @ 65C for 15 minutes to inactivate enzyme (phosphotase).
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| Gene | 10x Buffer | DNA | Phosphotase
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| Base Vector | 5.6uL | 50.0uL | 1.0uL
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| NOTE: Total volume in dephosphorylation = 56.6uL
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| 2. Run RXN model on Calhoun (super computer).
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| 3. Dilute base vector culture, allow to grow, then miniprep, then streak plates and make base vector glycerol stocks.
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| 4. Autoclave dishes
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| 5. Ligation: Follow table below. When ligation is complete, incubate products @16C for 1 hour. Heat inactivate DNA ligase (enzyme) @ 65C for 15 minutes.
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| Reagent | Volume in uL
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| 10x Buffer | 2.0uL
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| H20 | 20.0uL
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| Vector |
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| DNA |
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| T4 DNA Ligase | 1.0uL
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| 6. Ligation product Transformations: Transform the ligation products to TOP10 E. Coli cells. Allow growth for 2 hours.
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