Team:Paris/July 29
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The ladder used was the 100 bp ladder from New England Biolabs. | The ladder used was the 100 bp ladder from New England Biolabs. | ||
- | |||
- | + | ||
+ | === List of the digestion === | ||
+ | |||
+ | [[Image:KR000081.jpg|thumb|]] | ||
{| border="1" | {| border="1" | ||
Line 58: | Line 60: | ||
! 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 | ||
|- | |- | ||
Line 69: | Line 71: | ||
! 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 | ||
|- | |- | ||
Line 80: | Line 82: | ||
! 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 | ||
|- | |- | ||
Line 91: | Line 93: | ||
! 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 | ||
|- | |- | ||
Line 102: | Line 104: | ||
! 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 | ||
|- | |- | ||
Line 113: | Line 115: | ||
! 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 | ||
|- | |- | ||
Line 124: | Line 126: | ||
! 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 | ||
|- | |- | ||
Line 135: | Line 137: | ||
! 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 | ||
|- | |- | ||
Line 146: | Line 148: | ||
! 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 | ||
|- | |- | ||
Line 157: | Line 159: | ||
! 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 | ||
|- | |- | ||
Line 168: | Line 170: | ||
! 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 | ||
|} | |} | ||
Line 175: | Line 177: | ||
==> '''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. | ==> '''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. | ||
+ | == Transformations == | ||
+ | === Protocol === | ||
- | == | + | ''Use of TOP10 Chemically competent cells'' |
+ | |||
+ | * Defroze competent cells on ice during 5' | ||
+ | * Add 5µl of DNA Ligation in 50µL of competent bacterias (or 1µL for the positive control puc19) | ||
+ | * Incubate 30' on ice | ||
+ | * Heat-shock the cells during 30" at 42°C without shaking | ||
+ | * Put 2' on ice | ||
+ | * Add 250µL of pre-warmed SOC medium (4°C) | ||
+ | * Incubate 1h at 37°C under shaking (225rpm) | ||
+ | * Spin at 5.000rpm during 30" | ||
+ | * Remove 150µL of supernatant | ||
+ | * Resuspent the pellet in the 150µL left | ||
+ | * Spread on adequated plates | ||
+ | * 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
|