Team:Paris/July 29
From 2008.igem.org
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(→DNA digestion and purification) |
(→List of the Ligation Transformation) |
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! rowspan="1"| FV | ! rowspan="1"| FV | ||
! rowspan="1"| 2076 pb | ! rowspan="1"| 2076 pb | ||
- | ! rowspan="1"| | + | ! rowspan="1"| - |
- | ! rowspan="1"| | + | ! rowspan="1"| - |
! rowspan="1"| 10 | ! rowspan="1"| 10 | ||
|- | |- | ||
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! rowspan="1"| BV | ! rowspan="1"| BV | ||
! rowspan="1"| 2077 pb | ! rowspan="1"| 2077 pb | ||
- | ! rowspan="1"| | + | ! rowspan="1"| - |
- | ! rowspan="1"| | + | ! rowspan="1"| - |
! rowspan="1"| 11 | ! rowspan="1"| 11 | ||
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! rowspan="1"| BV | ! rowspan="1"| BV | ||
! rowspan="1"| 2222 pb | ! rowspan="1"| 2222 pb | ||
- | ! rowspan="1"| | + | ! rowspan="1"| - |
- | ! rowspan="1"| | + | ! rowspan="1"| - |
! rowspan="1"| 13 | ! rowspan="1"| 13 | ||
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! rowspan="1"| BV | ! rowspan="1"| BV | ||
! rowspan="1"| 2119 pb | ! rowspan="1"| 2119 pb | ||
- | ! rowspan="1"| | + | ! rowspan="1"| - |
- | ! rowspan="1"| | + | ! rowspan="1"| - |
! rowspan="1"| 12 | ! rowspan="1"| 12 | ||
|- | |- | ||
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! rowspan="1"| BI | ! rowspan="1"| BI | ||
! rowspan="1"| 707 pb | ! rowspan="1"| 707 pb | ||
- | ! rowspan="1"| | + | ! rowspan="1"| - |
- | ! rowspan="1"| | + | ! rowspan="1"| - |
! rowspan="1"| 14 | ! rowspan="1"| 14 | ||
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! rowspan="1"| BI | ! rowspan="1"| BI | ||
! rowspan="1"| 725 pb | ! rowspan="1"| 725 pb | ||
- | ! rowspan="1"| | + | ! rowspan="1"| - |
- | ! rowspan="1"| | + | ! rowspan="1"|- |
! rowspan="1"| 15 | ! rowspan="1"| 15 | ||
|- | |- | ||
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! rowspan="1"| FI | ! rowspan="1"| FI | ||
! rowspan="1"| 746 pb | ! rowspan="1"| 746 pb | ||
- | ! rowspan="1"| | + | ! rowspan="1"| - |
- | ! rowspan="1"| | + | ! rowspan="1"|- |
! rowspan="1"| 4 & 5 | ! rowspan="1"| 4 & 5 | ||
|- | |- | ||
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! rowspan="1"| BI | ! rowspan="1"| BI | ||
! rowspan="1"| 745 pb | ! rowspan="1"| 745 pb | ||
- | ! rowspan="1"| | + | ! rowspan="1"|- |
- | ! rowspan="1"| | + | ! rowspan="1"| - |
! rowspan="1"| 2 & 3 | ! rowspan="1"| 2 & 3 | ||
|- | |- | ||
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! rowspan="1"| FI | ! rowspan="1"| FI | ||
! rowspan="1"| 746 pb | ! rowspan="1"| 746 pb | ||
- | ! rowspan="1"| | + | ! rowspan="1"| - |
- | ! rowspan="1"| | + | ! rowspan="1"|- |
! rowspan="1"| 16 | ! rowspan="1"| 16 | ||
|- | |- | ||
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! rowspan="1"|FV | ! rowspan="1"|FV | ||
! rowspan="1"| 2079 pb | ! rowspan="1"| 2079 pb | ||
- | ! rowspan="1"| | + | ! rowspan="1"| - |
- | ! rowspan="1"| | + | ! rowspan="1"|- |
! rowspan="1"| 6 & 7 | ! rowspan="1"| 6 & 7 | ||
|- | |- | ||
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! rowspan="1"|BV | ! rowspan="1"|BV | ||
! rowspan="1"| 2080 pb | ! rowspan="1"| 2080 pb | ||
- | ! rowspan="1"| | + | ! rowspan="1"| - |
- | ! rowspan="1"| | + | ! rowspan="1"|- |
! rowspan="1"| 8 & 9 | ! rowspan="1"| 8 & 9 | ||
|} | |} | ||
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=== Protocol === | === Protocol === | ||
- | + | ''Use of TOP10 Chemically competent cells'' | |
- | + | ||
* Defroze competent cells on ice during 5' | * Defroze competent cells on ice during 5' | ||
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* Spread on adequated plates | * Spread on adequated plates | ||
* Incubate O/N at 37°C | * Incubate O/N at 37°C | ||
+ | |||
+ | |||
+ | === List of the Ligation Transformation === | ||
+ | |||
+ | |||
+ | {| border="1" | ||
+ | |align="center"|'''Name''' | ||
+ | |align="center"|'''Description''' | ||
+ | |align="center"|'''Antibio''' | ||
+ | |- | ||
+ | |colspan="3"|'''Ligation ''' | ||
+ | |- | ||
+ | |align="center"|L100 | ||
+ | |align="center"|rbs TetR - ECFP<br>D110 (BV) - D130 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L101 | ||
+ | |align="center"|rbs TetR - GFP tripart<br>D110 (BV) - D131 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L102 | ||
+ | |align="center"|Strong rbs - YFP<br>D129 (BV) - D118 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L103 | ||
+ | |align="center"|Strong rbs - mRFP<br>D129 (BV) - D122 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L104 | ||
+ | |align="center"|Strong rbs - lasR activator<br>D129 (BV) - D114 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L105 | ||
+ | |align="center"|Strong promoter - ECFP<br>D123 (BV) - D130 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L106 | ||
+ | |align="center"|Strong promoter - gfp Tripart<br>D123 (BV) - D131 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L107 | ||
+ | |align="center"|Strongest promoter - ECFP<br>D103 (BV) - D130 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L108 n°2 (the right one) | ||
+ | |align="center"|Strong promoter - gfp Tripart<br>D103 (BV) - D131 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L109 n°1 | ||
+ | |align="center"|Strong promoter - ecfp<br>D124 (BV) - D130 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L109 n°2 | ||
+ | |align="center"|Strong promoter - ecfp<br>D124 (BV) - D130 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L110 | ||
+ | |align="center"|Medium promoter - gfp Tripart<br>D124 (BV) - D131 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L111 | ||
+ | |align="center"|Weak promoter - ECFP<br>D104 (BV) - D130 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L112 | ||
+ | |align="center"|Weak promoter - gfp tripart<br>D104 (BV) - D131 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L113 | ||
+ | |align="center"|AracpBAD - ecfp<br>D126 (BV) - D130 (BI) | ||
+ | |align="center"|Kan | ||
+ | |- | ||
+ | |align="center"|L114 | ||
+ | |align="center"|AracpBAD - gfp tripart<br>D126 (BV) - D131 (BI) | ||
+ | |align="center"|Kan | ||
+ | |- | ||
+ | |align="center"|L115 | ||
+ | |align="center"|pLas - ECFP<br>D105 (BV) - D130 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L116 | ||
+ | |align="center"|pLas - gfp Tripart<br>D105 (BV) - D131 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L117 | ||
+ | |align="center"|yfp - Double Terminator<br>D117 (FI) - D125 (FV) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L118 | ||
+ | |align="center"|rfp - Double Terminator<br>D121 (FI) - D125 (FV) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L119 | ||
+ | |align="center"|lasR activator + LVA - Double Terminator<br>D113 (FI) - D125 (FV) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L120 | ||
+ | |align="center"|tetR repressible promoter - ECFP<br>D106 (BV) - D130 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L121 | ||
+ | |align="center"|Strong promoter - gfp tripart<br>D106 (BV) - D131 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L122 | ||
+ | |align="center"|RBS-lasI - ecfp<br>D107 (BV) - D130 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L123 | ||
+ | |align="center"|RBS lasI - gfp Tripart<br>D107 (BV) - D131 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L124 | ||
+ | |align="center"|Strongest RBS - mRFP<br>D102 (BV) - D122 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L125 | ||
+ | |align="center"|Strongest RBS - yfp<br>D102 (BV) - D118 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L126 | ||
+ | |align="center"| | ||
+ | Strongest RBS (1)- LacR activator (+LVA)<br>D102 (BV) - D114 (BI) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|L127 | ||
+ | |align="center"|gfp (1)- Double terminator<br>D119 (FI) - D125 (FV) | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |colspan ="3"|'''Controls''' | ||
+ | |- | ||
+ | |align="center"|C1 | ||
+ | |align="center"|D110 | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|C2 | ||
+ | |align="center"|D129 | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|C3 | ||
+ | |align="center"|D123 | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|C4 | ||
+ | |align="center"|D103 | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|C5 | ||
+ | |align="center"|D124 | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|C6 | ||
+ | |align="center"|D104 | ||
+ | |align="center"|Amp | ||
+ | |||
+ | |- | ||
+ | |align="center"|C7 | ||
+ | |align="center"|D126 | ||
+ | |align="center"|Kana | ||
+ | |- | ||
+ | |align="center"|C8 | ||
+ | |align="center"|D105 | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|C9 | ||
+ | |align="center"|D125 | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|C10 | ||
+ | |align="center"|D106 | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|C11 | ||
+ | |align="center"|D107 | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|C12 | ||
+ | |align="center"|D102 | ||
+ | |align="center"|Amp | ||
+ | |- | ||
+ | |align="center"|Positive control | ||
+ | |align="center"|puc19 | ||
+ | |align="center"|Amp | ||
+ | |} |
Latest revision as of 17:01, 13 August 2008
DNA digestion and purificationMix digestionfor each reaction (total volume : 50 µL)
ProtocolEach reaction was :
==> Unfortunately, there were not enough columms, so we took some columms from the QIAGEN MiniPrep kit, hoping that it will work with the QIAquick DNA Gel Extraction kit. Some of the samples were too voluminous, so we separated them into two tubes.
Each of the samples was then analysed by a 1,5% agarose gel:
The ladder used was the 100 bp ladder from New England Biolabs.
List of the digestion
==> Conclusion :The amount of DNA loaded was too low and the DNA ladder used was the wrong one. So this experiment has to be reconducted tomorrow. TransformationsProtocolUse of TOP10 Chemically competent cells
List of the Ligation Transformation
|