Team:Paris/July 29

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(Digestion of DNA)
(List of the Ligation Transformation)
 
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-
==Digestion of DNA==
+
{{Paris/Calendar_Links|July 28|July 30}}
-
for each reaction (total volume : 50 µL)
+
==DNA digestion and purification==
 +
 
 +
 
 +
=== Mix digestion ===
 +
 
 +
''for each reaction (total volume : 50 µL)''
* 20 µL of DNA (MiniPrep product)
* 20 µL of DNA (MiniPrep product)
* 2 µL of enzyme 1
* 2 µL of enzyme 1
* 2 µL of enzyme 2
* 2 µL of enzyme 2
* 5 µL of buffer 2 (10X)
* 5 µL of buffer 2 (10X)
 +
* 0,5 µL of BSA
* 20,5 µL water
* 20,5 µL water
-
Each reaction was incubated 2 hours at 37°C, then 10 minutes at 60-65°C (to inactivate the enzymes). 10 µL of loading dye (6X) were added to each of the 50 µL of digestion product. The whole samples were run in a 1,5% agarose gel (about 30 minutes at 100 W ; 2 x 30 µL per sample ; 30 µL per well). The bands of interest were then excised from the gel and the DNA was purified using the QIAquick DNA Gel Extraction kit (QIAGEN). 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. The elution of DNA was performed using 50 µL of water (after 10 minutes of incubation at 37°C).
+
 
 +
 
 +
=== Protocol ===
 +
 
 +
Each reaction was :
 +
* Incubate 2 hours at 37°C, then 10 minutes at 60-65°C (to inactivate the enzymes).  
 +
* Add 10 µL of loading dye (6X) to each of the 50 µL of digestion product.  
 +
* Run the whole samples in a '''1,5% agarose gel''' (about 30 minutes at 100 W ; 2 x 30 µL per sample ; 30 µL per well).  
 +
* Excise the bands of interest from the gel and the DNA was purified using the QIAquick DNA Gel Extraction kit (QIAGEN).  
 +
* The elution of DNA was performed using 50 µL of water (after 10 minutes of incubation at 37°C).
 +
 
 +
==> 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:
Each of the samples was then analysed by a 1,5% agarose gel:
Line 16: Line 35:
The ladder used was the 100 bp ladder from New England Biolabs.
The ladder used was the 100 bp ladder from New England Biolabs.
-
{|Name|Biobrick|enz 1|enz 2|Obs|Exp size|Mea size|Conc (ng/µL)|Gel|Band
+
 
 +
 
 +
=== List of the digestion ===
 +
 
 +
[[Image:KR000081.jpg|thumb|]]
 +
 
 +
{| border="1"
 +
|align=center|'''Name'''
 +
|align=center|'''BioBrick'''
 +
|align=center|'''Tube N°'''
 +
|align=center|'''Enz 1'''
 +
|align=center|'''Enz 2'''
 +
|align=center|'''Obs'''
 +
|align=center|'''Exp Size'''
 +
|align=center|'''Mea Size '''
 +
|align=center|'''Conc (ng/µl)'''
 +
|align=center|'''Band'''
 +
|-
 +
! rowspan="1" style="background: #ccccff;" | D101
 +
! rowspan="1" style="background: #ccccff;" | B0034
 +
! rowspan="1"|1
 +
! rowspan="1"| EcoRI
 +
! rowspan="1"| XbaI
 +
! rowspan="1"| FV
 +
! rowspan="1"| 2076 pb
 +
! rowspan="1"| -
 +
! rowspan="1"| -
 +
! rowspan="1"| 10
 +
|-
 +
! rowspan="1" style="background: #ccccff;" | D102
 +
! rowspan="1" style="background: #ccccff;" | B0034
 +
! rowspan="1"|1
 +
! rowspan="1"| SpeI
 +
! rowspan="1"| PstI
 +
! rowspan="1"| BV
 +
! rowspan="1"| 2077 pb
 +
! rowspan="1"| -
 +
! rowspan="1"| -
 +
! rowspan="1"| 11
 +
|-
 +
! rowspan="1" style="background: #ccccff;" | D105
 +
! rowspan="1" style="background: #ccccff;" | R0079
 +
! rowspan="1"|1
 +
! rowspan="1"| SpeI
 +
! rowspan="1"| PstI
 +
! rowspan="1"| BV
 +
! rowspan="1"| 2222 pb
 +
! rowspan="1"| -
 +
! rowspan="1"| -
 +
! rowspan="1"| 13
 +
|-
 +
! rowspan="1" style="background: #ccccff;" | D106
 +
! rowspan="1" style="background: #ccccff;" | R0040
 +
! rowspan="1"|1
 +
! rowspan="1"| SpeI
 +
! rowspan="1"| PstI
 +
! rowspan="1"| BV
 +
! rowspan="1"| 2119 pb
 +
! rowspan="1"| -
 +
! rowspan="1"| -
 +
! rowspan="1"| 12
 +
|-
 +
! rowspan="1" style="background: #ccccff;" | D108
 +
! rowspan="1" style="background: #ccccff;" | S03154
 +
! rowspan="1"|1
 +
! rowspan="1"| XbaI
 +
! rowspan="1"| PstI
 +
! rowspan="1"| BI
 +
! rowspan="1"| 707 pb
 +
! rowspan="1"| -
 +
! rowspan="1"| -
 +
! rowspan="1"| 14
 +
|-
 +
! rowspan="1" style="background: #ccccff;" | D111
 +
! rowspan="1" style="background: #ccccff;" | S03879
 +
! rowspan="1"|1
 +
! rowspan="1"| XbaI
 +
! rowspan="1"| PstI
 +
! rowspan="1"| BI
 +
! rowspan="1"| 725 pb
 +
! rowspan="1"| -
 +
! rowspan="1"|-
 +
! rowspan="1"| 15
 +
|-
 +
! rowspan="1" style="background: #ccccff;" | D115
 +
! rowspan="1" style="background: #ccccff;" | C0179
 +
! rowspan="1"|2
 +
! rowspan="1"| EcoRI
 +
! rowspan="1"| SpeI
 +
! rowspan="1"| FI
 +
! rowspan="1"| 746 pb
 +
! rowspan="1"| -
 +
! rowspan="1"|-
 +
! rowspan="1"| 4 & 5
 +
|-
 +
! rowspan="1" style="background: #ccccff;" | D116
 +
! rowspan="1" style="background: #ccccff;" | C0179
 +
! rowspan="1"|2
 +
! rowspan="1"| XbaI
 +
! rowspan="1"| PstI
 +
! rowspan="1"| BI
 +
! rowspan="1"| 745 pb
 +
! rowspan="1"|-
 +
! rowspan="1"| -
 +
! rowspan="1"| 2 & 3
 +
|-
 +
! rowspan="1" style="background: #ccccff;" | D117
 +
! rowspan="1" style="background: #ccccff;" | E0030
 +
! rowspan="1"|1
 +
! rowspan="1"| EcoRI
 +
! rowspan="1"| SpeI
 +
! rowspan="1"| FI
 +
! rowspan="1"| 746 pb
 +
! rowspan="1"| -
 +
! rowspan="1"|-
 +
! rowspan="1"| 16
 +
|-
 +
! rowspan="1" style="background: #ccccff;" | D128
 +
! rowspan="1" style="background: #ccccff;" | B0030
 +
! rowspan="1"|2
 +
! rowspan="1"|EcoRI
 +
! rowspan="1"|XbaI
 +
! rowspan="1"|FV
 +
! rowspan="1"| 2079 pb
 +
! rowspan="1"| -
 +
! rowspan="1"|-
 +
! rowspan="1"| 6 & 7
 +
|-
 +
! rowspan="1" style="background: #ccccff;" | D129
 +
! rowspan="1" style="background: #ccccff;" | B0030
 +
! rowspan="1"|2
 +
! rowspan="1"|SpeI
 +
! rowspan="1"|PstI
 +
! rowspan="1"|BV
 +
! rowspan="1"| 2080 pb
 +
! rowspan="1"| -
 +
! rowspan="1"|-
 +
! rowspan="1"| 8 & 9
 +
|}
 +
 
 +
==> '''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

← Yesterday

↓ Calendar ↑

Tomorrow →

Contents

DNA digestion and purification

Mix digestion

for each reaction (total volume : 50 µL)

  • 20 µL of DNA (MiniPrep product)
  • 2 µL of enzyme 1
  • 2 µL of enzyme 2
  • 5 µL of buffer 2 (10X)
  • 0,5 µL of BSA
  • 20,5 µL water


Protocol

Each reaction was :

  • Incubate 2 hours at 37°C, then 10 minutes at 60-65°C (to inactivate the enzymes).
  • Add 10 µL of loading dye (6X) to each of the 50 µL of digestion product.
  • Run the whole samples in a 1,5% agarose gel (about 30 minutes at 100 W ; 2 x 30 µL per sample ; 30 µL per well).
  • Excise the bands of interest from the gel and the DNA was purified using the QIAquick DNA Gel Extraction kit (QIAGEN).
  • The elution of DNA was performed using 50 µL of water (after 10 minutes of incubation at 37°C).

==> 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:

  • 2 µL of DNA
  • 3 µL of water
  • 1 µL of 6X loading dye

The ladder used was the 100 bp ladder from New England Biolabs.


List of the digestion

KR000081.jpg
Name BioBrick Tube N° Enz 1 Enz 2 Obs Exp Size Mea Size Conc (ng/µl) Band
D101 B0034 1 EcoRI XbaI FV 2076 pb - - 10
D102 B0034 1 SpeI PstI BV 2077 pb - - 11
D105 R0079 1 SpeI PstI BV 2222 pb - - 13
D106 R0040 1 SpeI PstI BV 2119 pb - - 12
D108 S03154 1 XbaI PstI BI 707 pb - - 14
D111 S03879 1 XbaI PstI BI 725 pb - - 15
D115 C0179 2 EcoRI SpeI FI 746 pb - - 4 & 5
D116 C0179 2 XbaI PstI BI 745 pb - - 2 & 3
D117 E0030 1 EcoRI SpeI FI 746 pb - - 16
D128 B0030 2 EcoRI XbaI FV 2079 pb - - 6 & 7
D129 B0030 2 SpeI PstI BV 2080 pb - - 8 & 9

==> 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

Name Description Antibio
Ligation
L100 rbs TetR - ECFP
D110 (BV) - D130 (BI)
Amp
L101 rbs TetR - GFP tripart
D110 (BV) - D131 (BI)
Amp
L102 Strong rbs - YFP
D129 (BV) - D118 (BI)
Amp
L103 Strong rbs - mRFP
D129 (BV) - D122 (BI)
Amp
L104 Strong rbs - lasR activator
D129 (BV) - D114 (BI)
Amp
L105 Strong promoter - ECFP
D123 (BV) - D130 (BI)
Amp
L106 Strong promoter - gfp Tripart
D123 (BV) - D131 (BI)
Amp
L107 Strongest promoter - ECFP
D103 (BV) - D130 (BI)
Amp
L108 n°2 (the right one) Strong promoter - gfp Tripart
D103 (BV) - D131 (BI)
Amp
L109 n°1 Strong promoter - ecfp
D124 (BV) - D130 (BI)
Amp
L109 n°2 Strong promoter - ecfp
D124 (BV) - D130 (BI)
Amp
L110 Medium promoter - gfp Tripart
D124 (BV) - D131 (BI)
Amp
L111 Weak promoter - ECFP
D104 (BV) - D130 (BI)
Amp
L112 Weak promoter - gfp tripart
D104 (BV) - D131 (BI)
Amp
L113 AracpBAD - ecfp
D126 (BV) - D130 (BI)
Kan
L114 AracpBAD - gfp tripart
D126 (BV) - D131 (BI)
Kan
L115 pLas - ECFP
D105 (BV) - D130 (BI)
Amp
L116 pLas - gfp Tripart
D105 (BV) - D131 (BI)
Amp
L117 yfp - Double Terminator
D117 (FI) - D125 (FV)
Amp
L118 rfp - Double Terminator
D121 (FI) - D125 (FV)
Amp
L119 lasR activator + LVA - Double Terminator
D113 (FI) - D125 (FV)
Amp
L120 tetR repressible promoter - ECFP
D106 (BV) - D130 (BI)
Amp
L121 Strong promoter - gfp tripart
D106 (BV) - D131 (BI)
Amp
L122 RBS-lasI - ecfp
D107 (BV) - D130 (BI)
Amp
L123 RBS lasI - gfp Tripart
D107 (BV) - D131 (BI)
Amp
L124 Strongest RBS - mRFP
D102 (BV) - D122 (BI)
Amp
L125 Strongest RBS - yfp
D102 (BV) - D118 (BI)
Amp
L126

Strongest RBS (1)- LacR activator (+LVA)
D102 (BV) - D114 (BI)

Amp
L127 gfp (1)- Double terminator
D119 (FI) - D125 (FV)
Amp
Controls
C1 D110 Amp
C2 D129 Amp
C3 D123 Amp
C4 D103 Amp
C5 D124 Amp
C6 D104 Amp
C7 D126 Kana
C8 D105 Amp
C9 D125 Amp
C10 D106 Amp
C11 D107 Amp
C12 D102 Amp
Positive control puc19 Amp