Team:Paris/August 12

From 2008.igem.org

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==='''Ligation'''===
==='''Ligation'''===
-
===='''List of ligations'''====
 
{| Border="2"
{| Border="2"
|align="center"|'''Ligation name'''
|align="center"|'''Ligation name'''
|align="center"|'''Insert name '''
|align="center"|'''Insert name '''
 +
|align="center"|'''Volume of insert µL'''
|align="center"|'''Vector name'''
|align="center"|'''Vector name'''
 +
|align="center"|'''Volume of Vector µL'''
|-
|-
|align="center"|L 139
|align="center"|L 139
|align="center"|D 140 (E0240 RBS +)
|align="center"|D 140 (E0240 RBS +)
 +
|align="center"| 3
|align="center"|D 144 (pSB3K3)
|align="center"|D 144 (pSB3K3)
 +
|align="center"| 3
|-
|-
|align="center"|L 140
|align="center"|L 140
|align="center"|D 141 (flhD)
|align="center"|D 141 (flhD)
 +
|align="center"| 3
|align="center"|D 145 (pSB1A2)
|align="center"|D 145 (pSB1A2)
 +
|align="center"| 3
|-
|-
|align="center"|L 141
|align="center"|L 141
|align="center"|D 142 (flhC)
|align="center"|D 142 (flhC)
 +
|align="center"| 3
|align="center"|D 145 (pSB1A2)
|align="center"|D 145 (pSB1A2)
 +
|align="center"| 3
|-
|-
|align="center"|L 142
|align="center"|L 142
|align="center"|D 143 (flhDC + promoter)
|align="center"|D 143 (flhDC + promoter)
 +
|align="center"| 3
|align="center"|D 145 (pSB1A2)
|align="center"|D 145 (pSB1A2)
 +
|align="center"| 3
|}
|}

Revision as of 15:10, 14 August 2008

← Yesterday

↓ Calendar ↑

Tomorrow →


Contents

Digestion and ligation of the PCR we did yesterday

Yesterday we amplified flhD, flhC, flhDC with its promoter and E040 RBS+ Before the digestion, we have to determine the DNA concentration of the templates.

Measurement of DNA concentration

We used the biophotometer. We put 5 µL of template DNA in 95 µL of water. The Blank was made with 5 µL of EB Buffer (the one that was used for the elution of the DNA) in 95 µL of water.

Template Concentration
(µg/µL)
PCR 130
E0240 RBS + 		0.44
PCR 131
flhD RBS - 		0.06
PCR 132
flhC RBS - 		0.12
PCR 133
flhDC with promoter 		0.08
MP 142
pSB3K3 		0.04
MP 122
pSB1A2 		0.2

Digestion

Protocol

Digestion name Template DNA Enzymes Volume of DNA
D 140 PCR 130 - E0240 RBS + PstI 5 µL
D 141 PCR 131 - flhD rbs - EcoRI-SpeI 5 µL
D 142 PCR 132 - flhC rbs - EcoRI-SpeI 5 µL
D 143 PCR 133 - flhDC + prom EcoRI-SpeI 5 µL
D 144 MP 142 - pSB3K3 EcoRI-SpeI 5 µL
D 145 MP122 - pSB1A2 EcoRI-SpeI 5 µL
  • X µL of Template DNA
  • Buffer (n°2) 10X : 3µL
  • BSA 100X : 0.3µL
  • Pure water qsp 30 µL
  • 1 µL of each enzyme
  • Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes).
  • Exclusively for D 144 : 2 µL of Antarctic Phosphatase was added, 30 min at 37°C then 5 min at 65°C.

Results of the digestion

    For the digestion after PCR, an electrophoresis is useless : we can not separate DNA fragments that have 10 bp of difference.

    Exclusively  for D 145 a gel extraction was made because there was two fragments, the standard protocol 
    was used (the picture is missing because we hadn't the USB key).
    The DNA purification after gel extraction was done according the standard protocol.

Ligation

Ligation name Insert name Volume of insert µL Vector name Volume of Vector µL
L 139 D 140 (E0240 RBS +) 3 D 144 (pSB3K3) 3
L 140 D 141 (flhD) 3 D 145 (pSB1A2) 3
L 141 D 142 (flhC) 3 D 145 (pSB1A2) 3
L 142 D 143 (flhDC + promoter) 3 D 145 (pSB1A2) 3

Minipreps: Plasmid extraction

  • Protocol (see # 3) Experiments done by QIAcube
  • List of Minipreps
Name Ligation Biobricks Description
MP144.1 L128.1 pFlgA-RFP
D132 (FI) - D136 (FV) 		Part icon regulatory.pngPart icon reporter.png
MP144.2 L128.2
MP144.3 L128.3
MP144.4 L128.4
MP145.1 L129.1 pFlgB-RFP
D133 (FI) - D136 (FV) 		Part icon regulatory.pngPart icon reporter.png
MP145.2 L129.2
MP145.6 L129.6
MP145.7 L129.7
MP146.1 L130.1 pFlhB-RFP
D134 (FI) - D136 (FV) 		Part icon regulatory.pngPart icon reporter.png
MP146.2 L130.2
MP146.7 L130.7
MP146.8 L130.8

Glycerol Stocks

  • List of Stocks
Strain Ligation Biobricks Description
S143.1 L128.1 pFlgA-RFP
D132 (FI) - D136 (FV) 		Part icon regulatory.pngPart icon reporter.png
S143.2 L128.2
S143.3 L128.3
S143.4 L128.4
S144.1 L129.1 pFlgB-RFP
D133 (FI) - D136 (FV) 		Part icon regulatory.pngPart icon reporter.png
S144.2 L129.2
S144.6 L129.6
S144.7 L129.7
S145.1 L130.1 pFlhB-RFP
D134 (FI) - D136 (FV) 		Part icon regulatory.pngPart icon reporter.png
S145.2 L130.2
S145.7 L130.7
S145.8 L130.8

Results of the transformations we did yesterday

Your results please Cyprien!!!???

Concentration of the MiniPreps

Miniprep Concentration (µg/mL) Ratio 260/280
MP144.1 127 1.67
MP144.2 166 1.66
MP144.3 163 1.57
MP144.4 154 1.63
MP145.1 136 1.67
MP145.2 151 1.63
MP145.6 163 1.67
MP145.7 188 1.57
MP146.1 298 1.70
MP146.2 142 1.64
MP146.7 170 1.52
MP146.8 143 1.55

New PCR screening with the right primers

Transformants with pFlgA, pFlgB and pFlhB cloned into J61002 are analysed by PCR but this time with the right primers: VF2 (O18) and VR (O19).

  • template: bacteria from glycerol stock
  • positive control: J61002 (with ptet-mRFP)
  • negative control: no template

Electrophoresis

  • 1% agarose gel
  • 10 µL of each sample loaded
well n° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
sample 1 kb DNA ladder positive control negative control
L128 (pFlgA)
L129 (pFlgB)
L130 (pFlhB)
100 bp DNA ladder
clone n° 1 2 3 4 1 2 6 7 1 2 7 8
red fluorescence
yes
yes
no no
yes
yes
no no
yes
no no no
expected size 1161 bp 0 bp
1339 bp
1339 bp
1338 bp
measured size 1,2 kb 0 kb 1,2 kb 1,2 kb
1,3 kb
1,3 kb
1,2 kb 0 kb
1,4 kb
1,4 kb
1,2 kb 1,2 kb 1,2 kb 1,2 kb
Screening-PCR-080812.JPG
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