Paris/October 4

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== Ligations ==
== Ligations ==
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{|
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|- style="background: #ccccff;"
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! colspan="5" style="background: #ccccff;" | Ligations
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|- style="background: #ccccff; text-align: center;"
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|width=5%| Number
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|width=20%| Insert
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|width=5%| Insert Volume (µL)
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|width=25%| Vector
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|width=5%| Vector Volume (µL)
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|width=35%| Comments
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|width=5%| Number of colonies
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |C19
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|
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|
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|D72
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|2
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|
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |C20
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|
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|
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|D73
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|2
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|
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |C21
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|
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|
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|D64
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|2
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|
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |L65
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|D56
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|10
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|D72
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|2
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|
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |L66
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|D75
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|10
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|D73
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|2
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|
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|
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |L67
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|D59
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|10
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|D64
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|2
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|
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|
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|}
== Culture of S28, strain having the [Frt-CmR-Frt] contruction ==
== Culture of S28, strain having the [Frt-CmR-Frt] contruction ==

Revision as of 15:48, 4 October 2007

Contents

Minipreps

  • from S49 = pKD46
  • pY-2P-IntC

Transformation of W121 and FR781 with pKD46

Houston we got a problem, the upstream construction for wanner lose the Chloramphenicol resistance

[Frt-CmR-Frt-pTet>>lox71-gfpTripart] (L58)

- First, we got an abnormally short length for the PCR with the wanner primers (about 1,7kb instead of 2,1kb; see October 2)
- Then i did a PCR on the same plasmid (L58) with the primers O18&O19. We got the same pattern of length (1,7kb and another one at about 3,5kb)
- To investigate the possibility to have lost 400kb in the CmR gene leading to the loss of resistence to Cm for the strain, i put some clones of L58 on plates with either Ampicilline or Chloramphenicol (10µg/mL).

> the clones grew on Amp but not on Cm...

it confirms the hypothesis of a loss inside the CmR gene.

> of course the results for the sequencing will give us the last word. They should come on Friday in late afternoon. We called the sequencing facility.

Digestion reactions

Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 Size Description
D79 L44.1 Xba1 Pst1
D80 L44.2 Xba1 Pst1
D81 L45.1 Xba1 Pst1
D82 L45.2 Xba1 Pst1
D83 L46.1 Xba1 Pst1
D84 L47.1 Xba1 Pst1
D85 L53.7 EcoRI SpeI
D86 L54.3 EcoRI SpeI
D87 L54.4 EcoRI SpeI
D88 L56.3 EcoRI SpeI
D89 L57.1 EcoRI SpeI
D90 L57.3 EcoRI SpeI
D91 MP14.1 EcoRI XbaI
D92 MP14.2 EcoRI XbaI
D93 MP14.1 Spe1 Pst1
D94 MP14.2 Spe1 Pst1

Ligations

Ligations
Number Insert Insert Volume (µL) Vector Vector Volume (µL) Comments Number of colonies
C19 D72 2
C20 D73 2
C21 D64 2
L65 D56 10 D72 2
L66 D75 10 D73 2
L67 D59 10 D64 2

Culture of S28, strain having the [Frt-CmR-Frt] contruction

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