"
Team:EPF-Lausanne/Notebook/Cloning Strategy
From 2009.igem.org
(→July) |
(→14.07.09) |
||
| Line 42: | Line 42: | ||
===14.07.09=== | ===14.07.09=== | ||
Primers designed for LOVTAP read-out and RBphP project: | Primers designed for LOVTAP read-out and RBphP project: | ||
| + | |||
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 | ||
gtttcttc*gaattcgcggccgcttctag*atgctggaggatatttgccctggtg | gtttcttc*gaattcgcggccgcttctag*atgctggaggatatttgccctggtg | ||
| + | |||
7.Reverse primer ppsR1 R.Palustris CGA009 | 7.Reverse primer ppsR1 R.Palustris CGA009 | ||
gtttcttc*ctgcagcggccgctactagta*ttactcatcggctccgtctccttc | gtttcttc*ctgcagcggccgctactagta*ttactcatcggctccgtctccttc | ||
| + | |||
8.Forward primer ppsR2 R.Palustris CGA009 | 8.Forward primer ppsR2 R.Palustris CGA009 | ||
gtttcttc*gaattcgcggccgcttctag*atggcgtcaaagtccgttcatgcc | gtttcttc*gaattcgcggccgcttctag*atggcgtcaaagtccgttcatgcc | ||
| + | |||
9.Reverse primer ppsR2 R.Palustris CGA009 | 9.Reverse primer ppsR2 R.Palustris CGA009 | ||
gtttcttc*ctgcagcggccgctactagta*tcaatcctctgcgtcgtctgagg | gtttcttc*ctgcagcggccgctactagta*tcaatcctctgcgtcgtctgagg | ||
| + | |||
10.Forward primer BrBphP Bradyrhizobium ORS278 | 10.Forward primer BrBphP Bradyrhizobium ORS278 | ||
gtttcttc*gaattcgcggccgcttctag*atgcccgttccgctgacgac | gtttcttc*gaattcgcggccgcttctag*atgcccgttccgctgacgac | ||
| + | |||
11.Reverse primer BrBphP Bradyrhizobium ORS278 | 11.Reverse primer BrBphP Bradyrhizobium ORS278 | ||
gtttcttc*ctgcagcggccgctactagta*tcactcctcgctctgcgagc | gtttcttc*ctgcagcggccgctactagta*tcactcctcgctctgcgagc | ||
| + | |||
12.Forward primer ppsR1 Bradyrhizobium ORS278 | 12.Forward primer ppsR1 Bradyrhizobium ORS278 | ||
gtttcttc*gaattcgcggccgcttctag*atgagggcgttcagagctcc | gtttcttc*gaattcgcggccgcttctag*atgagggcgttcagagctcc | ||
| + | |||
13.Reverse primer ppsR1 Bradyrhizobium ORS278 | 13.Reverse primer ppsR1 Bradyrhizobium ORS278 | ||
gtttcttc*ctgcagcggccgctactagta*ctattccaactgactgtcttcttcgc | gtttcttc*ctgcagcggccgctactagta*ctattccaactgactgtcttcttcgc | ||
| + | |||
14.Forward primer ppsR2 Bradyrhizobium ORS278 | 14.Forward primer ppsR2 Bradyrhizobium ORS278 | ||
gtttcttc*gaattcgcggccgcttctag*atggccgagtttcacggtccac | gtttcttc*gaattcgcggccgcttctag*atggccgagtttcacggtccac | ||
| + | |||
15.Reverse primer ppsR2 Bradyrhizobium ORS278 | 15.Reverse primer ppsR2 Bradyrhizobium ORS278 | ||
gtttcttc*ctgcagcggccgctactagta*ctagctccccttttcggtttcctc | gtttcttc*ctgcagcggccgctactagta*ctagctccccttttcggtttcctc | ||
Revision as of 15:08, 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
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
