"
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
