From 2008.igem.org
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| | |RBS+ | | |RBS+ |
| | |O141_O140 | | |O141_O140 |
| | + | |MG1655 |
| | + | |- style="text-align: center;" |
| | + | |PCR_131 |
| | + | |flhD RBS- |
| | + | |O132_O133 |
| | + | |MG1655 |
| | + | |- style="text-align: center;" |
| | + | |PCR_132 |
| | + | |flhC RBS- |
| | + | |O130_O131 |
| | + | |MG1655 |
| | + | |- style="text-align: center;" |
| | + | |PCR_133 |
| | + | |pflhDC |
| | + | |O110_O131 |
| | + | |MG1655 |
| | + | |- style="text-align: center;" |
| | + | |PCR_134 |
| | + | |pflhDC |
| | + | |O111_O131 |
| | |MG1655 | | |MG1655 |
| | |} | | |} |
Revision as of 12:17, 12 August 2008
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Transformation
Digestion
PCR
We performed PCR on to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.
PCR amplification
Protocol
| Number
| Name
| Sequence
| Length
| Comments
|
| O110
| FlhDC-Uri-F
| GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTTGTATGTGCGTGTAGTGACGAGTACAG
| 58
| Don't amplify the both OmpR binding site
|
| O111
| FlhDC-Total-F
| GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTCATTTTTGCTTGCTAGCGTACGGAAAA
| 58
| Amplify the both OmpR binding site
|
| O113
| FlhDC(nu)-R
| GTTTCTTCCTGCAGCGGCCGCTACTAGTACAGAATAACCAACTTTATTTTTATG
| 54
| Don't amplify the natural rbs of FlhD (only promoter)
|
| O124
| Oligo-pfliL-Forward-TRO
| TCGAATTCGCGGCCGCTTCTAGAGCAAGGGCGTGTAACAGGCAAC
| 45
|
|
| O125
| Oligo-pfliL-Reverse-TRO
| TCCTGCAGCGGCCGCTACTAGTAGTCATGTGTTGCGGTCTTCCTGTG
| 47
|
|
| O130
| Gene-FlhC-F
| GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAGTGAAAAAAGCATTGTTCAGG
| 53
|
|
| O131
| Gene-FlhC-R-TRO
| GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAAACAGCCTGTACTCTCTGTTCATCC
| 60
|
|
| O132
| Gene-FlhD-F
| GTTTCTTCGAATTCGCGGCCGCTTCTAGATGCATACCTCCGAGTTGCTGAAAC
| 53
| Don't amplify the natural rbs of FlhD
|
| O133
| Gene-FlhD-R-TRO
| GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAGGCCCTTTTCTTGCGCAGCGCTTCT
| 60
|
|
| O140
| PlasTest_GFP_Rv
| CCCTGCAGTTAATTAATATAAACGCAGAAAGGCCAC
| 36
| Amplify E0240 with a PacI and a PstI restriction site at the end
|
| O141
| PTst_GFPrbs+_F
| GCCTGCAGTCACACAGGAAAGTACTAGATGC
| 31
| Amplify E0240 with the promoter and a PstI restriction site at the beginning
|
- Preparation of the templates :
Resuspension of 1 colony in 100µl of water.
For each sample,
1 µl dNTP
10 µl Buffer Phusion 5x
2,5 µl Oligo_F
2,5 µl Oligo_R
1µl template
1 µl Phusion
50 µl qsp H2O (33µl)
| Name
| genes
| Oligo
| templates
|
| PCR_130
| RBS+
| O141_O140
| MG1655
|
| PCR_131
| flhD RBS-
| O132_O133
| MG1655
|
| PCR_132
| flhC RBS-
| O130_O131
| MG1655
|
| PCR_133
| pflhDC
| O110_O131
| MG1655
|
| PCR_134
| pflhDC
| O111_O131
| MG1655
|
Culture of ligation transformants
- 4 clones of each transformation were cultured in 7,5 mL LB + ampicilline. The colonies picked up were the rest of those already picked up from the transformation plate (for the PCR screening of August 8).
- 37°C overnight
| Ligation
| L128
| L129
| L130
|
| Name
| pFlgA
| pFlgB
| pFlhB
|
| Clone N°
| 1
| 2
| 3
| 4
| 1
| 2
| 6
| 7
| 1
| 2
| 7
| 8
|
| Red fluorescence
| yes
| yes
| no
| no
| yes
| yes
| no
| no
| yes
| no
| no
| no
|
|
"
