Team:Paris/August 16

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=Construction of pLas-TetR-GFP tripart & rbs-LasR-dble ter=
=Construction of pLas-TetR-GFP tripart & rbs-LasR-dble ter=
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=='''Transformation'''==
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==Transformation==
[[Team:Paris/Notebook/Protocols#Transformation |Transformation protocol]]
[[Team:Paris/Notebook/Protocols#Transformation |Transformation protocol]]

Revision as of 13:07, 30 August 2008

← Yesterday

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Contents

Analysis of the transformation we did yesterday

L143, L144, T1 and T2 showed no colonies. The positive control with pUC19 worked well.
We suppose that the ligation did not work. We will do it again today.

Construction of OmpR*+RBS and EnvZ*+RBS: Ligations

Cleaning of the DNA after the digestion

We used the QIAcube to wash the DNA, following the standard protocol.

Measure of DNA concentration of the digestion products

We used the biophotometer.
Settings:

  • 10 µL of template DNA in 50 µL of pure water
  • Blank : 10 µL of EB buffer in 50 µL of water.
Digestion name What's in ? DNA concentration (ng/µL)
D 158 OmpR* 12
D 159 EnvZ* 7
D 102 B0034 16

List of ligations

  • We ligated the DNA following the standard protocol.
  • T5 is the autoligation control for L148 and L149.
Ligation name Insert name Vinsert µL Vector name VVector µL
L 148 (Amp) D 158 (OmpR*) 4 D 102 (B0034) 3
L 149 (Amp) D 159 (EnvZ) 7 D 102 (B0034) 3

Ligation

Cleaning of the DNA after the digestion

We used the QIAcube to wash the DNA, following the standard protocol.

Measure of DNA concentration of the digestion products

We used the biophotometer.
Settings:

  • 10 µL of template DNA in 50 µL of pure water
  • Blank : 10 µL of EB buffer in 50 µL of water.
Digestion name What's in ? DNA concentration (ng/µL)
D 149 pfliL 2
D 150 pfliL 3
D 151 pSB3K3 12
D 152 pSB3K3 20
D 153 gene flhDC 7
D 154 gene flhDC 12
D 155 pflhDC 16
D 136 j61002 12
D 145 pSB1A2 21

List of ligations

  • We ligated the DNA following the standard protocol.
  • T1, T2, T3 and T4 are the autoligation controls for L 143, L144, L145 and L147
Ligation name Insert name Vinsert µL Vector name VVector µL
L 143 (Kan) D 149 (pfliL) 8 D 137 (pSB3K3) 4
L 144 (Kan) D 150 (pfliL) 5 D 152 (pSB3K3) 2.5
L 145 (Amp) D 153 (g flhDC) 10 D 145 (pSB1A2) 2.5
L 146 (Amp) D 154 (g flhDC) 5 D 145 (pSB1A2) 2.5
L 147 (Amp) D 155 (p flhDC) 1 D 136 (J61002) 4

Construction of pLas-TetR-GFP tripart & rbs-LasR-dble ter

Transformation

Transformation protocol

Name Description Antibio
Ligations
L150 D105(BV) - D146(BI)
pLas - strongest rbs-TetR-GFP tripart 		Amp
L151 D147(FI) - D125 (FV)
strongest rbs-LasR activator with LVA - Double terminator 		Kan
Controls
C1 D105 Amp
C2 D125 Kan
Positive Control pUC19 Amp


Digestion check from yesterday

16-08-08.png

Protocol Digestion

Digestion name Plasmid Description Miniprep used Comments
MP3 B0015 (double terminator B0010-B0012) - FV 4 No bands
D164 MP101 promoter J23101 - BV 1 Good expected sized bands
D161 MP104 PTet (Tet promoter) - FI 1 Good expected sized band
D162 MP114 TetR - BI 1 Good expected sized bands
D163 MP143 gfp generator - BV 2 Good expected sized bands
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