Team:Paris/August 12

From 2008.igem.org

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==Digestion of the PCR we did yesterday==
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Yesterday we amplified ''flhD'', ''flhC'', ''flhDC'' with its promoter and ''RBS+''
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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.
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{| Border="2"
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|align="center"|'''Template'''
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|align="center"|'''Concentration '''<br>(µg/µL)
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|-
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|align="center"| PCR 130 <br> E0240 RBS +
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|align="center"| 0.44
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|-
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|align="center"| PCR 131 <br> ''flhD'' RBS -
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|align="center"| 0.06
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|-
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|align="center"| PCR 132 <br> ''flhC'' RBS -
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|align="center"| 0.12
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|-
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|align="center"| PCR 133 <br> ''flhDC'' with promoter
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|align="center"| 0.08
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|-
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|align="center"| MP 142 <br> pSB3K3
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|align="center"| 0.04
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|-
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|align="center"| MP 122 <br> pSB1A2
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|align="center"| 0.2
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|-
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|}
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==='''Digestion'''===
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====Protocol====
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{| Border="2"
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|align="center"|'''Digestion name'''
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|align="center"|'''Template DNA'''
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|align="center"|''' Enzymes '''
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|align="center"|'''Volume of DNA'''
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|-
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|align="center"|D 137
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|align="center"|
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|align="center"| PstI
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|align="center"|25 µL
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|-
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|align="center"|D 138
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|align="center"|MP 143 - E0240
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|align="center"|EcoRI-SpeI
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|align="center"|6.25 µL
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|}
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* X µL of Template DNA
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* Buffer (n°2) 10X : 3µL
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* BSA 100X : 0.3µL
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* Pure water qsp 30 µL
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* 1 µL of each enzyme
 +
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* Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes).
 +
 +
====Results of the digestion====
 +
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[[Image:KR000131.jpg|thumb|Result of the digestion to build the measurement plasmid]]
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 +
'''Electrophoresis settings'''
 +
* Gel : 1 % agar
 +
* 4µL template DNA for D137
 +
* All the digestion product for D138 because we have to separate two products, the backbone measures 2056 bp and the insert we want to extract is 899 bp long.
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* 10µL QuickLoad DNA ladder 1 kb
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 +
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{| border="1"
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|align="center"|'''Name'''
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|align="center"|'''Band'''
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|align="center"|'''Expected size'''
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|align="center"|'''Measured size'''
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|-
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|align="center"|D 137
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|align="center"|2
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|style="background: #cbff7B"|<center>2727 bp</center>
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|align="center"|3000 bp
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|-
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|align="center"|D 138
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|align="center"|3&4
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|style="background: #cbff7B"|<center>899 bp</center>
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|align="center"|~1000 bp
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|}
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==='''Gel extraction and DNA purification'''===
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To extract the biobrick E0240 from the gel, we used the standard protocol [[Team:Paris/Notebook/Protocols#Extraction|number 8]].<br>
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To purify the DNA we used the standard protocol [[Team:Paris/Notebook/Protocols#Purification_.28Kit_Promega.29|number 10]]
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The ligation will be done [[Team:Paris/August_9|tomorrow]].
== Minipreps: Plasmid extraction==
== Minipreps: Plasmid extraction==

Revision as of 14:03, 14 August 2008

← Yesterday

↓ Calendar ↑

Tomorrow →


Contents

Digestion of the PCR we did yesterday

Yesterday we amplified flhD, flhC, flhDC with its promoter and 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 137 PstI 25 µL
D 138 MP 143 - E0240 EcoRI-SpeI 6.25 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).

Results of the digestion

Result of the digestion to build the measurement plasmid

Electrophoresis settings

  • Gel : 1 % agar
  • 4µL template DNA for D137
  • All the digestion product for D138 because we have to separate two products, the backbone measures 2056 bp and the insert we want to extract is 899 bp long.
  • 10µL QuickLoad DNA ladder 1 kb


Name Band Expected size Measured size
D 137 2
2727 bp
3000 bp
D 138 3&4
899 bp
~1000 bp

Gel extraction and DNA purification

To extract the biobrick E0240 from the gel, we used the standard protocol number 8.
To purify the DNA we used the standard protocol number 10

The ligation will be done tomorrow.

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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