Team:Paris/August 21

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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

Ligation

Protocol

Ligation Name Vector Name Volume Vector (µL) Insert Volume Insert (µL)
L160 D166 3.63 D132 2.03
L161 D167 4.00 D132 2.01
Control L160 D166 3.63 - -
Control L161 D167 4.00 - -


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
  • positive control: S158 (pSB3K3)
  • negative control: no template

Electrophoresis

Gel n°1 (E0240 screening)
Gel n°2 (FlhDC+promotor screening)
  • 1% agarose gel
  • 10 µL loaded
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
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 0,3 kb


Results:

  • The clone of E0240 (S159.1) always have several bands amplified by PCR. It might contain different plasmids.
  • The clone of FlhDC+promotor (S161.1) don't have the correct size band. It also doesn't have the insert in the plasmid.
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