Team:Paris/August 22

From 2008.igem.org

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[[Team:Paris/Notebook/Protocols#Transformation |Protocol]]
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Revision as of 17:01, 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

Table reminder

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
Control L156 D161 1.25 none Vector autoligation control Kana
L157 D125.2 B0015 15 3 D162 tetR 11 4 tetR-B0015 Kana
Control L157 D125.2 3 none Vector autoligation control Kana
L166 D185 RBS B0032 21 2 D182 tetR 10 6.5 RBS B0032 - tetR Amp
Control L166 D185 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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