Minnesota/10 July 2008

From 2008.igem.org

Latest revision as of 20:33, 23 July 2008

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

Dephosphorylate Base Vector Diagram

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 Buffer H20 Insert DNA LAMBDAcI DNA Dephosphor. Base V. T4 DNA Ligase pro-LAMBDAcI-B.V. 4.0uL 0 promoter 1.0uL 6.0uL 10.0uL 1.0uL LAMBDAcI-term-B.V. 4.0uL 0 terminator 1.0uL 6.0uL 10.0uL 1.0uL Control 4.0uL 7.0uL 0 0 10.0uL 1.0uL

6. Ligation product Transformations: Transform the ligation products to TOP10 E. Coli cells. Allow growth for 2 hours. Following growth, 200 uL of all four ligation reactions and a control were plated on Kanamycin LB and were incubated at 37 degrees overnight.

7. Ordered Primers: Ordered primers for possible use in real-time PCR.

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