Team:Paris/August 15

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* Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes).
* Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes).
 +
Then 10°C overnight.
=='''Digestion'''==
=='''Digestion'''==

Revision as of 14:25, 17 August 2008

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Contents

For Kok-Phen

I did some digestions, ligations and screening for you. I tried to build : RBS (B0034) + OmpR* and RBS (B0034) + EnvZ*

Protocol

Digestion name Template DNA Enzymes Volume of DNA
D 158 MP 155.1 - OmpR* XbaI - PstI 5 µL
D 159 MP 156.1 - EnvZ* XbaI - PstI 5 µL
D 102 MP 100 - B0034 SpeI - PstI 5 µL
  • X µL of Template DNA
  • Buffer (n°2) 10X : 3µL
  • BSA 100X : 0.3µL
  • Pure water qsp 30 µL
  • 1 µL of each enzyme
  • Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes). Then 10°C overnight.

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 
We managed to amplify flhDC !

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 
We managed to amplify the promoter of flhDC

Digestions

After having succeded in amplifying the promoter and the gene of flhDC, we decided to clone it into a plasmid. The first step is the digestion.

Protocol

Digestion name Template DNA Enzymes Volume of DNA
D 153 PCR 138 - g flhDC EcoRI - PstI 5 µL
D 154 PCR 140 - g flhDC EcoRI - PstI 5 µL
D 155 PCR 139 - p flhDC EcoRI - PstI 5 µL
D 145 MP122 - pSB1A2 EcoRI-SpeI 5 µL
D 136 MP103 - J61002 EcoRI-SpeI 5 µL
  • X µL of Template DNA
  • Buffer (n°2) 10X : 3µL
  • BSA 100X : 0.3µL
  • Pure water qsp 30 µL
  • 1 µL of each enzyme
  • Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes). 
Then 10°C overnight.

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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