Team:Paris/August 22

From 2008.igem.org

(Difference between revisions)
(→Construction of pFlgA - GFP Generator)
(→Analysis of the transformant of FlhDC+promotor)
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*1 µL SpeI
*1 µL SpeI
*22,7 µL water
*22,7 µL water
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|colspan="4"|PCR
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|colspan="3"|digestion
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|'''sample'''
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|1 kb DNA ladder
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|MP143
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|negative control
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|MP160.1
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|MP143
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|100 bp DNA ladder
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='''Construction of pFlgA - GFP Generator'''=
='''Construction of pFlgA - GFP Generator'''=

Revision as of 16:29, 22 August 2008

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Contents

Analysis of the transformant of FlhDC+promotor

KR000219.jpg
  • Analysis of the plasmids MP160.1 and MP160.2 (FlhDC+promotor in pSB1A2)
  • Control: MP143 (GFP generator in pSB1A2)

PCR

PCR screening programm
elongation time: 1 min 30
total volume reaction (25 µL)

  • 12,5 µL Quick load PCR Mix 2X
  • 0,5 µL O18
  • 0,5 µL O19
  • 1 µL DNA
  • 10,5 µL water

Digestion

total volume reaction (30 µL)

  • 2 µL DNA
  • 3 µL buffer 2 10X
  • 0,3 µL BSA 100X
  • 1 µL XbaI
  • 1 µL SpeI
  • 22,7 µL water
well n° 1 2 3 4 5 6 7 8 9
method PCR digestion
sample 1 kb DNA ladder MP143 negative control MP160.1 MP160.2 MP143 MP160.1 MP160.2 100 bp DNA ladder
expected size
measured size

Construction of pFlgA - GFP Generator

Aim : Construction of "pFlgA-RBS-GFP-dbl ter" (pFlgA-E0430/E0432) Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png

Digestion

Digestion

Protocol Digestion

Name Template DNA Description Vol MP (µl) Vol H2O (µl) Enzymes
D168 MP143.1 RBS - GFP - term (FV) 7.25 17.45 EcoRI and XbaI

Gel Extraction

Protocol [[Image:KR00019b.jpg| thumb|Gel Extraction of D168]

Well 1 2 3 4 5 6
Sample 100 pb ladder MP143.1 no sample D168 no sample
Expected size (pb)
Measured size (pb)

Measurement of the concentration of D168 purified

Protocol (it's same that for Miniprep)

Digestion Name Concentration (µg/mL) Ratio 260/280
D168

Ligation

Protocol

Ligation Name Vector Name Volume Vector (µL) Insert Volume Insert (µL)
L164 D168 D132
Control L164 D168 - -

Construction of pFlgA - YFP tripart (+/- LVA)

Aim : Construction of "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432) Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png

Transformation of the ligations we did yesterday

Protocol

Ligation Name Description Antibio
L160 D166(FV) + D132 (FI) Amp
Control L160 D166 Amp
L161 D167(FV) + D1132 (FI) Amp
Control L161 D167 Amp

Construction for synchronization

Transformation of the ligations we did yesterday

Protocol

Ligation Name Description Antibio
L158 D110(BV) + D131 (BI) Amp
T L158 D110 Amp
L159 D125(FV)+D109(FI) Kan
T L159 D125 Kan

Ligation

Protocol

Ligation Name Description Vector Name Volume vector (µL) Insert Name Volume insert(µL)
L162 rbs-lasI + gfp generator
Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png 		D107 (BV) 10 D163 (BI) 3.46
T L162 autoligation control D107 (BV) 10
L163 rbs-TetR + gfp generator
Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png 		D110 (BV) 2.5 D163 (BI) 3.44
T L163 autoligation control D110 (BV) 2.5

Transformation

Protocol

Ligation Name Description Antibio
L162 D107(BV) + D163 (BI) Amp
T L162 D107 Amp
L163 D110(BV)+D163(BI) Amp
T L163 D110 Amp
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