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Team:EPF-Lausanne/Notebook/Cloning Strategy
From 2009.igem.org
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:
gtttcttc*gaattcgcggccgcttctag*atgctggaggatatttgccctggtg
7.Reverse primer ppsR1 R.Palustris CGA009:
gtttcttc*ctgcagcggccgctactagta*ttactcatcggctccgtctccttc
8.Forward primer ppsR2 R.Palustris CGA009:
gtttcttc*gaattcgcggccgcttctag*atggcgtcaaagtccgttcatgcc
9.Reverse primer ppsR2 R.Palustris CGA009:
gtttcttc*ctgcagcggccgctactagta*tcaatcctctgcgtcgtctgagg
10.Forward primer BrBphP Bradyrhizobium ORS278:
gtttcttc*gaattcgcggccgcttctag*atgcccgttccgctgacgac
11.Reverse primer BrBphP Bradyrhizobium ORS278:
gtttcttc*ctgcagcggccgctactagta*tcactcctcgctctgcgagc
12.Forward primer ppsR1 Bradyrhizobium ORS278:
gtttcttc*gaattcgcggccgcttctag*atgagggcgttcagagctcc
13.Reverse primer ppsR1 Bradyrhizobium ORS278:
gtttcttc*ctgcagcggccgctactagta*ctattccaactgactgtcttcttcgc
14.Forward primer ppsR2 Bradyrhizobium ORS278:
gtttcttc*gaattcgcggccgcttctag*atggccgagtttcacggtccac
15.Reverse primer ppsR2 Bradyrhizobium ORS278:
gtttcttc*ctgcagcggccgctactagta*ctagctccccttttcggtttcctc
