Team:Paris/August 8

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Contents

Minipreps : Plasmid extraction

Extraction of pSB3K3 et E0240 in pSB1A2 plasmid from overnight bacteria culture using the QIAspin Miniprep Kit (QIAGEN) by QIACube.

  • Carried out 2 times (2 tubes)
name Biobrick plasmid
MP142 - pSB3K3
MP143 E0240 pSB1A2


Transformation results

Name Description Antibio Number of colonies Number of red fluorescent colonies
Ligation
L128 J61002-pFlgA
D136 (FV) - D132 (FI) 		Amp ~ 400 2
L129 J61002-pFlgB
D136 (FV) - D133 (FI) 		Amp 39 5
L130 J61002-pFlhB
D136 (FV) - D134 (FI) 		Amp ~ 1000 4 (but 3 are on the edge of the petri dishe)
L131 J61002-pFlhDC
D136 (FV) - D135 (FI) 		Amp 39 38
Control
Control 1 D136 Amp 0 0
Positive control pUC19 Amp 36 0

creening PCR of transformants

Preparation of the newly ammplified promoters

Electrophoresis of the PCR products made yesterday

Standart PCR to amplify pflgA, pflgB and pflhB(Gel1)
PCR with gradient to amplify pflhDC(Gel2)

Electrophoresis settings

  • Gel : 1.5 % agar
  • 3µL template DNA
  • 10µL QuickLoad DNA ladder 100 bp
Name Promotor Gel Band Expected size Measured size
PCR_124 pFlgA 1 2
261 pb
250 pb
PCR_125 pFlgB 1 3
261 pb
250 pb
PCR_126 pFlhB 1 4
260 pb
250 pb
PCR_127 pFlhDC 2 2 to 13
446 pb
nothing




Results

  • We have no results for pflhDC, wo don't know yet where is the problem. We will try with other conditions! (yet undetermined)
  • Concerning pflhB, pflgA and pflgB, the protocol seems to be very operational: we always have great results !
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