Team:Paris/August 22

From 2008.igem.org

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(→Transformation of ligations from August 20th)
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|'''Insert digestion'''
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|J23101 promoter-gfp generator
|Amp
|Amp
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|gfp generator
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Revision as of 16:41, 26 August 2008

← Yesterday

↓ Calendar ↑

Tomorrow →

Contents

Analysis of the transformant of FlhDC+promotor

  • 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

Incubation 2h55 at 37°C and then 20 min at 65°C.

Electrophoresis

KR000219.jpg
  • 1% agarose gel
  • for PCR products: 10 µL loaded
  • for digestion products (30 µL): adding of 7 µL of loading blue and then 20 µL loaded on gel
well n° 1 2 3 4 5 6 7 8 9
method PCR digestion
sample 1 kb DNA ladder positive control MP143 negative control MP160.1 MP160.2 MP143 MP160.1 MP160.2 100 bp DNA ladder
expected size 1114 bp 1403 bp 876 bp (E0240)
2079 (pSB1A2) 		1165 bp (FlhDC+promotor)
2079 bp (pSB1A2)
measured size 1 kb
3 kb 		0,3 kb
1,8 kb 		0,3 kb
1,8 kb 		0,9 kb
2 kb 		2 kb 2 kb


Results: The clones tested didn't have the insert.

Construction of pFlgA - GFP Generator

Aim : Construction of "pFlgA-RBS-GFP-dbl ter" (pFlgA-E0240) 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

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) 2 940
Measured size (pb) 5.000 3 000

Measurement of the concentration of D168 purified

Protocol (it's same that for Miniprep)

Digestion Name Concentration (µg/mL) Ratio 260/280
D168 24 2.15

Ligation

Protocol

Ligation Name Vector Name Volume Vector (µL) Insert Volume Insert (µL)
L164 D168 1.67 D132 2.04
Control L164 D168 1.67 - -

Construction for FIFO

Aim : Construction of pFlgA - YFP tripart (+/- LVA) "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)
pFlgA - YFP tripart (LVA-) 		Amp
TL160 D166
YFP tripart (LVA-) 		Amp
L161 D167 (FV) + D1132 (FI)
pFlgA - YFP tripart (LVA+) 		Amp
TL161 D167
YFP tripart (LVA+) 		Amp

Construction for synchronization

Transformation of the ligations we did yesterday

Protocol

Ligation Name Description Antibio
L158 D110 (BV) + D131 (BI) Amp
Control L158 D110 Amp
L159 D125 (FV) + D109 (FI) Kan
Control 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
Control 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
Control L163 autoligation control D110 (BV) 2.5 - -

Transformation

Protocol

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


Promoter characterization plasmids

Transformation of ligations from August 20th

Top 10 cells were used


Protocol

a changer

Ligation name Vector digestion Vector description Vector conc. ug/mL Vector volume Insert digestion Insert description Insert conc. ug/mL Insert volume Product description Antibiotic
L155 D164 J23101 promoter 3 16 D163 gfp generator 14 8 J23101 promoter-gfp generator Amp
L156 D161 pTet promoter 39 1.25 D163 gfp generator 14 5 pTet promoter-gfp generator Kana
D161 1.25 none Vector autoligation control Kana
L157 D125.2 B0015 15 3 D162 11 4 tetR tetR-B0015 Kana
Control L157 D125.2 3 Vector autoligation control Kana
L166 D185 RBS B0032 21 2 D182 tetR 10 6.5 RBS B0032 - tetR Amp
Control L166 D185 RBS B0032 2 none Vector autoligation control Amp
L167 D181 pTet 1 14 D184 gfp generator 14 3 gfp generator - pTet Amp
Control L167 D181 pTet 14 none Vector autoligation control Amp
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