Team:Paris/August 15

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(→PCR amplification of flhDC and its promoter)
(→PCR amplification of flhDC and its promoter)
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=='''PCR amplification of flhDC and its promoter'''==
=='''PCR amplification of flhDC and its promoter'''==
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As we did not manage to amplofy flhDC or its promoter alone, we decided to amplify both flhDC and its promoter. From the big amplicon, we will try to get the promoter alone.
 
==='''List of PCRs'''===
==='''List of PCRs'''===

Revision as of 18:00, 16 August 2008

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Contents

Transformation of the ligations we did yesterday

We transformed L 143, L 144 and the negative controls T1 and T2, using Invitrogen's TOP10 chemically competent cells standard protocol.

PCR amplification of flhDC and its promoter

List of PCRs

Name of the PCR PCR 136 PCR 137 PCR 138 PCR 136' PCR 137' PCR 138' PCR 139 PCR 140
Forward primer O 110 O 111 O 131 O 110 O 111 O 131 O 110 O 131
Reverse primer O 113 O 113 O 132 O 113 O 113 O 132 O 113 O 131
Template DNA MG 1655 MG 1655 MG 1655 xx xx xx PCR 130 PCR 130

Protocol

We followed the standard protocol of amplification in Two steps. PCR program used : PHUSION2

Results

Settings Gel 1%

Gel 1%
  • Ladder 1 kb 10 µL
  • 4µL Template DNA + 2µL Loading Blue
  • 1 % Agar


PCR Name What's in ? Well Expected size Measured size
PCR_138 gene flhDC 2 992 bp 1000 bp
PCR_140 gene flhDC 3 992 bp 1000 bp
PCR_138' gene flhDC 4 X X
PCR_133 gene flhDC + promoter 5 1227 pb 1200 bp


Settings Gel 2%

Gel 2%
  • Ladder 100 bp 10 µL
  • 4µL Template DNA + 2µL Loading Blue
  • 2 % Agar


PCR Name What's in ? Well Expected size Measured size
PCR_136 pflhDC (URI) 2 282 bp X
PCR_137 pflhDC 3 428 bp X
PCR_139 pflhDC (URI) 4 282 bp ~ 300 bp
PCR_136' pflhDC (URI) 5 X X
PCR_137' pflhDC 6 X X

Digestions

Digestion

Measurement of concentration of minipreps

standard protocol

Digestion Miniprep used Concentration (µg/mL) ratio 260/280
D146 MP148.2 123 1.58
D147 MP153.3 113 1.68

Digestion

Protocol Digestion

Ligation

Protocol Ligation

Ligation name Insert Vector
L150 D146 (strongest rbs-TetR-GFP tripart) D105 (pLas)
L151 D147 (strongest rbs-LasR activator with LVA) D125 (Double terminator)
Control 1 / D105
Control 2 / D125
Positive Control Puc19

Analysis of yesterday PCR screening

Gel 1 KR000163.jpg Gel 2 KR000162.jpg Gel 3 KR000164.jpg


Starting the construction of the Promoter characterization plasmid

Digestion

Measurement of concentration of minipreps

standard protocol

Plasmid Miniprep Concentration (µg/mL) ratio 260/280
MP3 3 152 1.43
MP3 4 775 1.21
MP101 1 317 1.66
MP101 2 389 1.36
MP101 4 209 1.76
MP104 1 173 1.32
MP104 3 43 1.85
MP104 4 52 1.66
MP114 1 173 1.75
MP114 2 263 1.43
MP143 1 133 1.55
MP143 2 132 1.70

Digestion

Protocol Digestion

Plasmid Description Miniprep used Enzymes
MP3 B0015 (double terminator B0010-B0012) 4 EcoRI and XbaI
MP114 TetR 1 EcoRI and SpeI
MP104 PTet (Tet promoter) 1 SpeI and PstI
MP101 promoter J23101 1 SpeI and PstI
MP143 gfp generator 2 SpeI and PstI
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