Minnesota/10 July 2008

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| NOTE: Total volume in dephosphorylation = 56.6uL
| NOTE: Total volume in dephosphorylation = 56.6uL

Revision as of 19:20, 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 complete, 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


6. Ligation product Transformations: Transform the ligation products to TOP10 E. Coli cells. Allow growth for 2 hours.
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