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Team:EPF-Lausanne/Notebook/Cloning Strategy
From 2009.igem.org
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1.Forward primer Trp promoter: | 1.Forward primer Trp promoter: | ||
| - | + | :gtttcttc gaattcgcggccgcttctagagtggcaaatattctgaaatgagctgttgacaattaatcatcgaactagttaactagtacgc | |
| - | gtttcttc gaattcgcggccgcttctagagtggcaaatattctgaaatgagctgttgacaattaatcatcgaactagttaactagtacgc | + | |
2.Reverse primer Trp promoter: | 2.Reverse primer Trp promoter: | ||
| - | + | :ctagctagctaggtcgataccctttttacgtgaacttgcgtactagttaactagttcgatgattaattgtca | |
| - | ctagctagctaggtcgataccctttttacgtgaacttgcgtactagttaactagttcgatgattaattgtca | + | |
3.1st Forward primer Inverter TetR: | 3.1st Forward primer Inverter TetR: | ||
| - | + | :aatcatcgaactagttaactagtacgcaagttcacgtaaaaagggtatcgacaaagaggagaaatactagatgtcc | |
| - | aatcatcgaactagttaactagtacgcaagttcacgtaaaaagggtatcgacaaagaggagaaatactagatgtcc | + | |
4.2nd Forward primer Inverter TetR: | 4.2nd Forward primer Inverter TetR: | ||
| - | + | :gtttcttcgaattcgcggccgcttctagagtggcaaatattctgaaatgagctgttgacaattaatcatcgaactagttaactagta | |
| - | gtttcttcgaattcgcggccgcttctagagtggcaaatattctgaaatgagctgttgacaattaatcatcgaactagttaactagta | + | |
5.Reverse Primer Inverter TetR: | 5.Reverse Primer Inverter TetR: | ||
| - | + | :ctagctagctag tttctcctctttctctagtagtgc | |
| - | ctagctagctag tttctcctctttctctagtagtgc | + | |
6.Forward primer ppsR1 R.Palustris CGA009: | 6.Forward primer ppsR1 R.Palustris CGA009: | ||
| - | + | :gtttcttcgaattcgcggccgcttctagatgctggaggatatttgccctggtg | |
| - | + | ||
7.Reverse primer ppsR1 R.Palustris CGA009: | 7.Reverse primer ppsR1 R.Palustris CGA009: | ||
| - | + | :gtttcttcctgcagcggccgctactagtattactcatcggctccgtctccttc | |
| - | + | ||
8.Forward primer ppsR2 R.Palustris CGA009: | 8.Forward primer ppsR2 R.Palustris CGA009: | ||
| - | + | :gtttcttcgaattcgcggccgcttctagatggcgtcaaagtccgttcatgcc | |
| - | + | ||
9.Reverse primer ppsR2 R.Palustris CGA009: | 9.Reverse primer ppsR2 R.Palustris CGA009: | ||
| - | + | :gtttcttcctgcagcggccgctactagtatcaatcctctgcgtcgtctgagg | |
| - | + | ||
10.Forward primer BrBphP Bradyrhizobium ORS278: | 10.Forward primer BrBphP Bradyrhizobium ORS278: | ||
| - | + | :gtttcttcgaattcgcggccgcttctagatgcccgttccgctgacgac | |
| - | + | ||
11.Reverse primer BrBphP Bradyrhizobium ORS278: | 11.Reverse primer BrBphP Bradyrhizobium ORS278: | ||
| - | + | gtttcttcctgcagcggccgctactagtatcactcctcgctctgcgagc | |
| - | + | ||
12.Forward primer ppsR1 Bradyrhizobium ORS278: | 12.Forward primer ppsR1 Bradyrhizobium ORS278: | ||
| - | + | :gtttcttcgaattcgcggccgcttctagatgagggcgttcagagctcc | |
| - | + | ||
13.Reverse primer ppsR1 Bradyrhizobium ORS278: | 13.Reverse primer ppsR1 Bradyrhizobium ORS278: | ||
| - | + | :gtttcttcctgcagcggccgctactagtactattccaactgactgtcttcttcgc | |
| - | + | ||
14.Forward primer ppsR2 Bradyrhizobium ORS278: | 14.Forward primer ppsR2 Bradyrhizobium ORS278: | ||
| - | + | :gtttcttcgaattcgcggccgcttctagatggccgagtttcacggtccac | |
| - | + | ||
15.Reverse primer ppsR2 Bradyrhizobium ORS278: | 15.Reverse primer ppsR2 Bradyrhizobium ORS278: | ||
| - | + | :gtttcttcctgcagcggccgctactagtactagctccccttttcggtttcctc | |
| - | + | ||
===15.07.09=== | ===15.07.09=== | ||
Revision as of 15:11, 14 July 2009
Contents |
Cloning strategy
July
06.07.09
Four forward primers were designed to amplify:
1.Promoter T7, RBS, CBP and LOVTAP:
- gtttcttcgaattcgcggccgcttctagagtaatacgactcactataggggaattgtg
2.RBS, CBP and LOVTAP:
- gtttcttcgaattcgcggccgcttctagagtgtttaactttaagaaggag
3.CBP and LOVTAP:
- gtttcttcgaattcgcggccgcttctagatgaagcgacgatggaaaaagaatttcatag
4.LOVTAP:
- gtttcttcgaattcgcggccgcttctagatgctactacacttgaacgtattgagaagaac
One reverse primer were designed:
- gtttcttcctgcagcggccgctactagtatcaatcgcttttcagcaacacctcttc
The recipient IGEM part have been chosen: [http://partsregistry.org/partsdb/get_part.cgi?part=BBa_B0010 BBa_B0010], well 13D in the received kit plate 1
07.07.09
To design plasmids : software Vector NTI
08.07.09
Inducible LOVTAP biobrick strategy
09.07.09
Partial digestion strategy.
10.07.09
13.07.09
Restriction enzymes on [http://www.neb.com/nebecomm/products/category1.asp?#2 Biolabs website] and [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.asp clevage oligonucleotides]
TRP promoter biobrick strategy
14.07.09
Primers designed for LOVTAP read-out and RBphP project:
1.Forward primer Trp promoter:
- gtttcttc gaattcgcggccgcttctagagtggcaaatattctgaaatgagctgttgacaattaatcatcgaactagttaactagtacgc
2.Reverse primer Trp promoter:
- ctagctagctaggtcgataccctttttacgtgaacttgcgtactagttaactagttcgatgattaattgtca
3.1st Forward primer Inverter TetR:
- aatcatcgaactagttaactagtacgcaagttcacgtaaaaagggtatcgacaaagaggagaaatactagatgtcc
4.2nd Forward primer Inverter TetR:
- gtttcttcgaattcgcggccgcttctagagtggcaaatattctgaaatgagctgttgacaattaatcatcgaactagttaactagta
5.Reverse Primer Inverter TetR:
- ctagctagctag tttctcctctttctctagtagtgc
6.Forward primer ppsR1 R.Palustris CGA009:
- gtttcttcgaattcgcggccgcttctagatgctggaggatatttgccctggtg
7.Reverse primer ppsR1 R.Palustris CGA009:
- gtttcttcctgcagcggccgctactagtattactcatcggctccgtctccttc
8.Forward primer ppsR2 R.Palustris CGA009:
- gtttcttcgaattcgcggccgcttctagatggcgtcaaagtccgttcatgcc
9.Reverse primer ppsR2 R.Palustris CGA009:
- gtttcttcctgcagcggccgctactagtatcaatcctctgcgtcgtctgagg
10.Forward primer BrBphP Bradyrhizobium ORS278:
- gtttcttcgaattcgcggccgcttctagatgcccgttccgctgacgac
11.Reverse primer BrBphP Bradyrhizobium ORS278: gtttcttcctgcagcggccgctactagtatcactcctcgctctgcgagc
12.Forward primer ppsR1 Bradyrhizobium ORS278:
- gtttcttcgaattcgcggccgcttctagatgagggcgttcagagctcc
13.Reverse primer ppsR1 Bradyrhizobium ORS278:
- gtttcttcctgcagcggccgctactagtactattccaactgactgtcttcttcgc
14.Forward primer ppsR2 Bradyrhizobium ORS278:
- gtttcttcgaattcgcggccgcttctagatggccgagtttcacggtccac
15.Reverse primer ppsR2 Bradyrhizobium ORS278:
- gtttcttcctgcagcggccgctactagtactagctccccttttcggtttcctc
