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Minnesota/10 July 2008
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| 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). |
| Gene | 10x Buffer | DNA | Phosphotase |
|---|---|---|---|
| Base Vector | 5.6uL | 50.0uL | 1.0uL |
| NOTE: Total volume in dephosphorylation = 56.6uL |
| 2. Run RXN model on Calhoun (super computer). |
| 3. Dilute base vector culture, allow to grow, then miniprep, then streak plates and make base vector glycerol stocks. |
| 4. Autoclave dishes |
| 5. Ligation: Follow table below. When ligation is completed, incubate products @16C for 1 hour. Heat inactivate DNA ligase (enzyme) @ 65C for 15 minutes. |
| Reagent | Volume in uL |
|---|---|
| 10x Buffer | 2.0uL |
| H20 | 20.0uL |
| Vector | |
| DNA | |
| T4 DNA Ligase | 1.0uL |
