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.
|
| Primer | Sequence
|
| F Mcherry for modification | gaattcgcggccgcttctagatggtgagcaagggcgagg
|
| R Mcherry for modification | TATAAACGCAGAAAGGCCCACCC
|
| R LAMcI for realtime PCR | GGTTGTGCTTACCCATCTCTCC
|
| R p22mnt for realtime PCR | ACTCGCTCTGCTCATCGGCG
|
| R p22cII for realtime PCR | CAATCTACAGTGGTGTCGTGCC
|
| R GFP for realtime PCR | GAATGTTTCCATCTTCTTTAAAATC
|
| R mCherry for realtime PCR | GTGATGAACTTCGAGGACGGCG
|
"
