Team:Paris/August 21

From 2008.igem.org

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Revision as of 14:10, 21 August 2008

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Contents

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

Digestion

Gel Extraction

Protocol [[Image:KR00019b.jpg| thumb|Gel Extraction of D166-D167]

Well 1 2 3 4 5 6 7 8 9 10 11 12
Sample 1kb ladder MP165.1 MP166.1 no sample D166 no sample D167 no sample 100pb ladder no sample
Expected size (pb) 2 957 2 996 2942 2981
Measured size (pb) 3 000 3 000

Screening of the cloning of E0240 and FlhDC+promotor

We obtained single colonies with the dilution 100.
13 clones were analysed by PCR

PCR screening

reaction mixture (25 µL)

  • 12,5 µL Quick load PCR Mixture 2X
  • 0,5 µL O18
  • 0,5 µL O19
  • 11,5 µL water

PCR screening programm

  • elongation time: 1 min 30
  • primers: O18 and O19
Gel n°1 (E0240 in pSB3K3)
well n° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
sample 1 kb DNA ladder positive control negative control S159.1 100 bp DNA ladder
colonie n° 1 2 3 4 5 6 7 8 9 10 11 12 13
expected size 1192 bp
measured size 1,5 kb
1,1 kb
0,6 kb 		1,5 kb
1,1 kb
0,6 kb 		1,5 kb
1,1 kb
0,6 kb 		1,5 kb
1,1 kb
0,6 kb 		1,5 kb
1,1 kb
0,6 kb 		1,5 kb
1,1 kb
0,6 kb 		1,5 kb
1,1 kb
0,6 kb 		1,5 kb
1,1 kb
0,6 kb 		1,5 kb
1,1 kb
0,6 kb 		1,5 kb
1,1 kb
0,6 kb 		1,5 kb
1,1 kb
0,6 kb 		1,5 kb
1,1 kb
0,6 kb 		1,5 kb
1,1 kb
0,6 kb
Gel n°2 (FlhDC+promotor in pSB1A2)
well n° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
sample 1 kb DNA ladder positive control negative control S161.1 100 bp DNA ladder
colonie n° 1 2 3 4 5 6 7 8 9 10 11 12 13
expected size 1403 bp
measured size
Gel n°1 (E0240 screening)
Gel n°2 (FlhDC+promotor screening)
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