Team:Paris/August 14

From 2008.igem.org

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T1 and T2 are the autoligation controls for L143 and L144.
T1 and T2 are the autoligation controls for L143 and L144.
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==PCR screening of cloning of E0240, flhD, flhC and pflhDC+flhDC==
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*PCR screening programm
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*elongation time: 1 min 30
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*total volume reaction: 50 microL
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*primers: O18 & O19
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*positive control 1: E0240
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*positive control 2: pSB3K3
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*negative control: no template

Revision as of 14:58, 15 August 2008

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Contents

Results of the PCR of the wonderful promoter of fliL

Amplification of pfliL


Electrophoresis settings :

  • Gel 1.5% agar
  • Ladder 100 bp
  • Volume of template : 3 µL


Name Promotor Well Expected size Measured size
PCR_135 pfliL 2 197 bp
~ 200 bp
PCR_135' Negative Control 3 0 bp
0 bp 
Remarks:
We observe that the bands are curved, we suppose that the wells were not very clean.
The size of fliL is good, we will digest it and ligate it today.

Digestion and Ligation of the wonderful promoter of fliL

As the primer used to amplify the promoter of fliL had only two nucleotides after the restriction sites, we tried the two digestions possible : EcoRI + SpeI and XbaI + PstI.

Digestion

Protocol :

Name Genes Water DNA Buffer n°2 10X BSA 100X Enz 1 Enz 2
D149 fliL 23 µL 2 µl 3 µl 0.30µl EcoRI SpeI
D150 fliL 23 µL 2 µl 3 µl 0.30µl XbaI PstI
D137 pSB3K3 23 µL 2 µl 3 µl 0.30µl EcoRI SpeI
D152 pSB3K3 23 µL 2 µl 3 µl 0.30µl XbaI PstI
  • Incubate 2h30 at 37°C
  • 20 min at 65°C to denaturate the enzymes.

Washing of the digestions

We washed the DNA following the standard protocol.

Ligation

Ligation name Insert name Volume of insert µL Vector name Volume of Vector µL
L 143 D 149 (pfliL) 2 D 137 (pSB3K3) 6
L 144 D 150 (pfliL) 2 D 152 (pSB3K3) 6
  • 2 µL Ligase Buffer 10X
  • X µg/µL vector
  • Y µg/µL insert
  • Pure water qsp 20 µL
  • 1 µL T4 ligase
  • O/N at 16°C

T1 and T2 are the autoligation controls for L143 and L144.

PCR screening of cloning of E0240, flhD, flhC and pflhDC+flhDC

  • PCR screening programm
  • elongation time: 1 min 30
  • total volume reaction: 50 microL
  • primers: O18 & O19
  • positive control 1: E0240
  • positive control 2: pSB3K3
  • negative control: no template
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