Paris/July 27
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We will use the protocol of the keio collection: [http://ecoli.naist.jp/gb6/Resources/deletion/Del_construct.html] | We will use the protocol of the keio collection: [http://ecoli.naist.jp/gb6/Resources/deletion/Del_construct.html] | ||
| - | * 20nt of upstream region of kan gene, 5'-ATTCCGGGGATCCGTCGACC-3' (P1) | + | * 20nt of upstream region of kan gene, |
| + | 5'-ATTCCGGGGATCCGTCGACC-3' (P1) | ||
| - | * 20nt of kan downstream sequence, 5'-TGTAGGCTGGAGCTGCTTCG-3' (P2) | + | * 20nt of kan downstream sequence, |
| + | 5'-TGTAGGCTGGAGCTGCTTCG-3' (P2) | ||
===DapA deletion=== | ===DapA deletion=== | ||
| - | *50nt DapA upstream region 5'-CATACCAAACGTACCATTGAGACACTTGTTTGCACAGAGGATGGCCCATG-3' (P3) | + | *50nt DapA upstream region |
| - | *50nt DapA downstream region (reverse complement) 5'-AAGCCATCAAATCTCCCTAAACTTTACAGCAAACCGGCATGCTTAAGCGC-3' (P4) | + | 5'-CATACCAAACGTACCATTGAGACACTTGTTTGCACAGAGGATGGCCCATG-3' (P3) |
| + | *50nt DapA downstream region (reverse complement) | ||
| + | 5'-AAGCCATCAAATCTCCCTAAACTTTACAGCAAACCGGCATGCTTAAGCGC-3' (P4) | ||
| - | * dDapA-F CATACCAAACGTACCATTGAGACACTTGTTTGCACAGAGGATGGCCCATGATTCCGGGGATCCGTCGACC (P3 + P1) | + | * dDapA-F |
| + | CATACCAAACGTACCATTGAGACACTTGTTTGCACAGAGGATGGCCCATGATTCCGGGGATCCGTCGACC (P3 + P1) | ||
| - | * dDapA-R AAGCCATCAAATCTCCCTAAACTTTACAGCAAACCGGCATGCTTAAGCGCTGTAGGCTGGAGCTGCTTCG (P6 + P2) | + | * dDapA-R |
| + | AAGCCATCAAATCTCCCTAAACTTTACAGCAAACCGGCATGCTTAAGCGCTGTAGGCTGGAGCTGCTTCG (P6 + P2) | ||
=== FtsZ deletion === | === FtsZ deletion === | ||
| - | *50nt FtsZ upstream region 5'-GACGATGATTACGGCCTCAGGCGACAGGCACAAATCGGAGAGAAACTATG-3' (P5) | + | *50nt FtsZ upstream region |
| - | *50nt FtsZ downstream region (reverse complement) 5'-GAAACCCAAATTCCAGTCAATTCTTAATCAGCTTGCTTACGCAGGAATGC-3' (P6) | + | 5'-GACGATGATTACGGCCTCAGGCGACAGGCACAAATCGGAGAGAAACTATG-3' (P5) |
| + | *50nt FtsZ downstream region (reverse complement) | ||
| + | 5'-GAAACCCAAATTCCAGTCAATTCTTAATCAGCTTGCTTACGCAGGAATGC-3' (P6) | ||
| - | *dFtsZ-F GACGATGATTACGGCCTCAGGCGACAGGCACAAATCGGAGAGAAACTATGATTCCGGGGATCCGTCGACC (P5 + P1) | + | *dFtsZ-F |
| + | GACGATGATTACGGCCTCAGGCGACAGGCACAAATCGGAGAGAAACTATGATTCCGGGGATCCGTCGACC (P5 + P1) | ||
| - | *dFtsZ-R GAAACCCAAATTCCAGTCAATTCTTAATCAGCTTGCTTACGCAGGAATGCTGTAGGCTGGAGCTGCTTCG (P6 + P2) | + | *dFtsZ-R |
| + | GAAACCCAAATTCCAGTCAATTCTTAATCAGCTTGCTTACGCAGGAATGCTGTAGGCTGGAGCTGCTTCG (P6 + P2) | ||
Revision as of 11:08, 27 July 2007
Contents |
PCR to perform
Perform the following PCR reactions
- P5 (then perform digestion rxn D18 & D20)
- P6 (then perform digestion reactions D19 & D21)
- P8
- P9
Repeat ligation reactions (same as 25/07/07). Also include digestion products D28 & D32
Oligo design for Wanner deletion
We were for now on not able to perform the deletion of DapA through transduction. So we will try the deletion through the Wanner method. We will also attempt the deletion of FtsZ.
We will use the protocol of the keio collection: [1]
- 20nt of upstream region of kan gene,
5'-ATTCCGGGGATCCGTCGACC-3' (P1)
- 20nt of kan downstream sequence,
5'-TGTAGGCTGGAGCTGCTTCG-3' (P2)
DapA deletion
- 50nt DapA upstream region
5'-CATACCAAACGTACCATTGAGACACTTGTTTGCACAGAGGATGGCCCATG-3' (P3)
- 50nt DapA downstream region (reverse complement)
5'-AAGCCATCAAATCTCCCTAAACTTTACAGCAAACCGGCATGCTTAAGCGC-3' (P4)
- dDapA-F
CATACCAAACGTACCATTGAGACACTTGTTTGCACAGAGGATGGCCCATGATTCCGGGGATCCGTCGACC (P3 + P1)
- dDapA-R
AAGCCATCAAATCTCCCTAAACTTTACAGCAAACCGGCATGCTTAAGCGCTGTAGGCTGGAGCTGCTTCG (P6 + P2)
FtsZ deletion
- 50nt FtsZ upstream region
5'-GACGATGATTACGGCCTCAGGCGACAGGCACAAATCGGAGAGAAACTATG-3' (P5)
- 50nt FtsZ downstream region (reverse complement)
5'-GAAACCCAAATTCCAGTCAATTCTTAATCAGCTTGCTTACGCAGGAATGC-3' (P6)
- dFtsZ-F
GACGATGATTACGGCCTCAGGCGACAGGCACAAATCGGAGAGAAACTATGATTCCGGGGATCCGTCGACC (P5 + P1)
- dFtsZ-R
GAAACCCAAATTCCAGTCAATTCTTAATCAGCTTGCTTACGCAGGAATGCTGTAGGCTGGAGCTGCTTCG (P6 + P2)
