Team:Paris/August 11

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Revision as of 17:54, 12 August 2008

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Contents

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

  • List of Oligos :
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.


  • Preparation of PCR mix :

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 E0240 RBS+ O141_O140 MP143
PCR_131 - O141_O140 Water
PCR_132 flhD RBS- O132_O133 MG1655
PCR_133 - O132_O133 Water
PCR_134 flhC RBS- O130_O131 MG1655
PCR_135 - O130_O131 Water
PCR_136 flhDC + prom O110_O131 MG1655
PCR_137 - O110_O131 Water
PCR_138 flhDC + prom O111_O131 MG1655
PCR_139 - O111_O131 Water
PCR_140 pfliL O124_O125 MG1655
PCR_141 - O124_O125 Water
PCR_142 pflhDC O110_O113 MG1655
PCR_143 - O110_O113 Water
PCR_144 pflhDC O111_O113 MG1655
PCR_145 - O111_O113 Water


  • Program PCR_Screening : Annealing 30" at 60°C - Time élongation 1'30" at 72°C - Number cycle : 30

PCR verification/Analysis

Analysis of PCR product (Gel 1)
Analysis of PCR product (Gel 2)

After the PCR :

  • 2*3µl have been analysed by electrophoresis
  • the other 44µl of PCR products have been purified by the Promega kit.


  • Electrophoresis

ladder : 10µl ladder 1 kb
samples : 3µl of PCR products + 2µl of Loading Dye
migration 30min at 100V, on a 1% agarose gel


  • Results :
Name Promotor Gel Band Expected size Measured size
PCR_130 E0240 Gel 1 2 876 bp
900 bp
PCR_131 Negative Control Gel 1 3 0 bp
0 bp
PCR_132 flhD RBS- Gel 1 4 351 bp
350 bp
PCR_133 Negative Control Gel 1 5 0 bp
0 bp
PCR_134 flhC RBS- Gel 1 6 579 bp
600 bp
PCR_135 Negative Control Gel 1 7 0 bp
0 bp
PCR_136 flhDC + prom Gel 2 2 1165 bp
1300 bp
PCR_137 Negative Control Gel 2 3 0 bp
0 bp
PCR_138 flhDC + prom Gel 2 4 1311 bp
2100 pb
PCR_139 Negative Control Gel 2 5 0 bp
0 pb


==> Conclusion : We observed the size expected for the PCR products, but not for pflhDC (PCR_138), is right. We hypothesis for PCR_138 that the size is longer that expected due to the aspecific fixation of Oligo O111 (upper to the real site).


  • Electrophoresis
Analysis of PCR product (Gel 3)

ladder : 10µl ladder 100 bp
samples : 3µl of PCR products + 2µl of Loading Dye
migration 30min at 100V, on a 2% agarose gel


  • Results :
Name Promotor Gel Band Expected size Measured size
PCR_140 pfliL Gel 3 2 124 bp
0 bp
PCR_141 Negative Control Gel 3 3 0 bp
0 bp
PCR_142 pflhDC Gel 3 4 223
0 bp
PCR_143 Negative Control Gel 3 5 0 bp
0 bp
PCR_144 pflhDC Gel 3 6 369
0 bp
PCR_145 Negative Control Gel 3 7 0 bp
0 bp


==> Conclusion : We need to do again the experience

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
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