Minnesota/10 July 2008
From 2008.igem.org
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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). | |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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| NOTE: Total volume in dephosphorylation = 56.6uL | | NOTE: Total volume in dephosphorylation = 56.6uL | ||
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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. | |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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Revision as of 19:24, 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).
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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.
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6. Ligation product Transformations: Transform the ligation products to TOP10 E. Coli cells. Allow growth for 2 hours. |