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Minnesota/10 July 2008
From 2008.igem.org
(Difference between revisions)
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|6. '''Ligation product Transformations''': Transform the ligation products to TOP10 E. Coli cells. Allow growth for 2 hours. | |6. '''Ligation product Transformations''': Transform the ligation products to TOP10 E. Coli cells. Allow growth for 2 hours. | ||
| + | |||
| + | |- | ||
| + | |7. '''Ordered Primers''': Ordered primers for possible use in real-time PCR. | ||
| + | |- | ||
| + | {|border="1" align="left" | ||
| + | |- | ||
| + | !| 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 | ||
| + | |- | ||
| + | |} | ||
|} | |} | ||
Revision as of 21:34, 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. | ||||||||||||
| 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 |
