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Minnesota/30 June 2008
From 2008.igem.org
| 1. Set up Sequencher: Need to determine what will be synthesized and research p22 mnt sequence. |
| 2. Repick miniprep plate #3 |
| 3. Redo glycerol stock #7 |
| 4. Discuss vectors |
| 5. Use Vector NTI: Choose the primer base pairs (essentially 20 b.p.'s long to lessen chance of hairpins or errors). Using Vector NTI, we designed primers which will allow us to add the RBS sequence, to the TetR promoter/lamba cI gene construct, through PCR rather than cloning. Since the primer sequence is approximately 8 base pairs long, use of the cloning procedure would be highly ineffective. In addition, we designed primers that will allow us to isolate the sequence of each of our dually repressed promoters, with only the prefix and suffix tails. |
- Designed Primers
| Sequence Name | Forward Primer | Reverse Primer |
|---|---|---|
| lambda cI | 5’ – TCACACAGGAAAGTACTAGATGAGCACAAAAAAGAAACC – 3’ (RBS tail)
5' - GAATTCGCGGCCGCTTCTAGAGTCACACAGGAAAGTACTAGATGAGC - 3' (Prefix tail) | 5’ – CTGCAGCGGCCGCTACTAGTATTATTAAGC – 3’ |
| LacI/λcI Promoter | 5' - GAATTCGCGGCCGCTTCTAGAGGC - 3' | 5' - CTGCAGCGGCCGCTACTAGTATGTGTGAAATTGTTATCCGC - 3' |
| TetR/p22 mnt Promoter | 5' - GAATTCGCGGCCGCTTCTAGAGTCCC - 3' | 5' - CTGCAGCGGCCGCTACTAGTAGTGCTCAGTATCTAGGTCACCG -3' |
