Team:Paris/August 7

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[[image:KR000117.jpg|thumb|PCR with gradient to amplify pflhDC]]
[[image:KR000117.jpg|thumb|PCR with gradient to amplify pflhDC]]
[[image:KR000116.jpg|thumb|Standart PCR to amplify pflgA, pflgB and pflhB]]
[[image:KR000116.jpg|thumb|Standart PCR to amplify pflgA, pflgB and pflhB]]
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==Transformations==
==Transformations==
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* Spread on adequated plates
* Spread on adequated plates
* Incubate O/N at 37°C
* Incubate O/N at 37°C
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=== List of the Ligation Transformation ===
=== List of the Ligation Transformation ===

Revision as of 13:43, 7 August 2008

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Contents

Result of the isolation of colonies

E0240 and pSB3K3 are ok : there is a lot of single colonies S120 and S121 : there is a problem, there is nothing on the plates. We have to check whether those strains are really resistant to Amp.

Results of the PCR we did last night

PCR with gradient to amplify pflhDC
Standart PCR to amplify pflgA, pflgB and pflhB




Transformations

Protocol

Use of TOP10 chemically competentcells

  • Defroze competent cells on ice during 5'
  • Add 5µl of DNA Ligation in 50µL of competent bacterias (or 1µL for the positive control puc19)
  • Incubate 30' on ice
  • Heat-shock the cells during 30" at 42°C without shaking
  • Put 2' on ice
  • Add 250µL of pre-warmed SOC medium (4°C)
  • Incubate 1h at 37°C under shaking (225rpm)
  • Spin at 5.000rpm during 30"
  • Remove 150µL of supernatant
  • Resuspent the pellet in the 150µL left
  • Spread on adequated plates
  • Incubate O/N at 37°C

List of the Ligation Transformation

Name Description Antibio
Ligation
L128 J61002-pFlgA
D136 (FV) - D132 (FI) 		Amp
L129 J61002-pFlgB
D136 (FV) - D133 (FI) 		Amp
L130 J61002-pFlhB
D136 (FV) - D134 (FI) 		Amp
L131 J61002-pFlhDC
D136 (FV) - D135 (FI) 		Amp
Control
Control 1 D136 Amp
Positive control pUC19 Amp
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