Team:Paris/July 25

From 2008.igem.org

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(Extraction of DNA from gel)
(list of Minipreps)
 
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-
== Extraction of DNA from gel ==
+
== DNA Extraction ==
-
Extraction of Minipreps DNA (MP100 --> MP122) from the gel.
+
* Use of Promega's protocol for the extraction of: gel n°1 --> 4 : (D100 --> D110 cl-1 ES))
 +
* Use of Qiagen's protocol for the extraction of: gel n°5 --> 8 : (D110 cl-2 ES --> D120))
-
* use of protocol of PROMEGA (gel n°1 --> 4 : (D100 --> D110 cl-1 ES))
+
* Analysis of the extraction success by electrophoresis on 2µl of the parts extracted.
-
* use of protocol of QIAGEN  (gel n°5 --> 8 : (D110 cl-2 ES --> D120))
+
-
== MiniPreps / Digestion ==
 
-
* Minipreps : Protocol of Promega has been done on all the clones cultivated on the 24th.
 
 +
=== Determination of DNA concentration by spectrophotometry ===
-
===Digestion Mix===
+
'''Conditions:'''
-
10µl of Miniprep <br>
 
-
12.5µl of water<br>
 
-
2.5µl of Buffer N°2<br>
 
-
0.25µl of BSA 100x<br>
 
-
1µl of enzyme 1<br>
 
-
1µl of enzyme 2<br>
 
 +
* 2µl of the DNA extracted / 98µl of pure water
-
===list of digestion===
+
* Reading against the adaptated blank (2µl of the elution's buffer / 98µl of pure water)
 +
 
 +
 
 +
 
 +
'''Results :'''
 +
 
 +
 
 +
-D100 --> D110 cl-1 ES : Absorbance ~20-50µg/ml ;  DO 260/280 = 1,5 ;  DO 260/230 = 0,0-0.20
 +
 
 +
-D110 cl-2 ES --> D120 : Absorbance ~0-10µg/ml
 +
 
 +
 
 +
 
 +
=== Determination of DNA concentration by electrophoresis ===
 +
 
 +
* to check for the samples from Qiagen's protocol, if the absence of detection of absorbance, is due to a problem of reading or a problem during the extraction, we realise an electrophoresis.
 +
 
 +
 
 +
==> conclusion : we don't succeed to detect DNA by spectrophometry or electrophoresis for the samples produced by Qiagen's protocol.
 +
 
 +
 
 +
==> we decide to repeat all the samples from the beginning, so we culture O/N at 37°c all the strains usefull for the experiments
 +
 
 +
== Culture ==
 +
 
 +
in 10ml LB with adaptated antibiotics, all the Biobricks used before (MP100 --> MP122)
 +
 
 +
* will be use to do minipreps
 +
 
 +
 
 +
== MiniPreps ==
 +
 
 +
 
 +
* Use of Promega's protocol on all the clones cultivated on the 24th.
 +
* Preparation of 50µl of minipreps in simple.
 +
 
 +
 
 +
 
 +
===list of Minipreps===
{| border="1"  
{| border="1"  
|align="center"|'''Name'''
|align="center"|'''Name'''
-
|align="center"|'''Miniprep'''
 
|align="center"|'''Biobrick'''
|align="center"|'''Biobrick'''
-
|align="center"|'''Enzyme 1'''
+
|align="center"|'''Description'''
-
|align="center"|'''Enzyme 2'''
+
-
|align="center"|'''Nber tube'''
+
|-
|-
-
|align="center"|D100
 
|align="center"|MP100
|align="center"|MP100
-
|align="center"|B0034
+
|align="center"|[http://partsregistry.org/Part:BBa_B0034 B0034]
-
|align="center"|XbaI
+
|align="center"|Strongest RBS (Efficiency = 1)
-
|align="center"|PstI
+
-
|align="center"|2
+
|-
|-
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|EcoRI
 
-
|align="center"|XbaI
 
-
|align="center"|1
 
-
|-
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|SpeI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|D116
 
|align="center"|MP116
|align="center"|MP116
-
|align="center"|J23100
+
|align="center"|[http://partsregistry.org/Part:BBa_J23100 J23100]
-
|align="center"|SpeI
+
|align="center"|Strong constitutive promoter in J61002
-
|align="center"|PstI
+
-
|align="center"|2
+
|-
|-
-
|align="center"|D120
 
|align="center"|MP120
|align="center"|MP120
-
|align="center"|B0030
+
|align="center"|[http://partsregistry.org/Part:BBa_B0030 B0030]
-
|align="center"|SpeI
+
|align="center"|Strong RBS (Efficiency = 0,6)
-
|align="center"|PstI
+
-
|align="center"|2
+
|-
|-
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|EcoRI
 
-
|align="center"|XbaI
 
-
|align="center"|1
 
-
|-
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|
 
-
|align="center"|XbaI
 
-
|align="center"|PstI
 
-
|align="center"|2
 
-
|-
 
-
|align="center"|D121
 
|align="center"|MP121
|align="center"|MP121
-
|align="center"|E0422
+
|align="center"|[http://partsregistry.org/Part:BBa_E0422 E0422]
-
|align="center"|XbaI
+
|align="center"|ECFP (RBS+LVA+Term)
-
|align="center"|PstI
+
-
|align="center"|1 !
+
-
|-
+
-
|align="center"|D122
+
-
|align="center"|MP122
+
-
|align="center"|E0840
+
-
|align="center"|XbaI
+
-
|align="center"|PstI
+
-
|align="center"|1 !
+
-
|}
+
-
 
+
-
 
+
-
=== Results of digestions : Electrophoresis ===
+
-
 
+
-
{| 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 Matrix'''
+
-
|align=center|'''Exp Size BB'''
+
-
|align=center|'''Mea Size Matrix'''
+
-
|align=center|'''Mea Size BB'''
+
-
|-
+
-
! rowspan="5" style="background: #ccccff;" | D100
+
-
! rowspan="5" style="background: #ccccff;" | B0034
+
-
|align=center|1
+
-
! rowspan="2"| XbaI
+
-
! rowspan="2"| PstI
+
-
! rowspan="2"| BI
+
-
! rowspan="2"| 2057 pb
+
-
! rowspan="2"| 34 pb
+
-
|align=center|
+
-
|align=center|
+
-
|-
+
-
|align=center|2
+
-
|align=center|
+
-
|align=center|
+
-
|-
+
-
|align=center|3
+
-
|align=center| EcoRI
+
-
|align=center| XbaI
+
-
|align=center| FV
+
-
|align=center| 2076 pb
+
-
|align=center| 15 pb
+
-
|align=center|
+
-
|align=center|
+
-
|-
+
-
|align=center|4
+
-
! rowspan="2"| SpeI
+
-
! rowspan="2"| PstI
+
-
! rowspan="2"| BV
+
-
! rowspan="2"| 2077 pb
+
-
! rowspan="2"| 14 pb
+
-
|align=center|
+
-
|align=center|
+
-
|-
+
-
|align=center|5
+
-
|align=center|
+
-
|align=center|
+
-
|-
+
-
! rowspan="1" style="background: #ccccff;"|D116
+
-
! rowspan="1" style="background: #ccccff;"|J23100
+
-
|align=center|1
+
-
! rowspan="1"| SpeI
+
-
! rowspan="1"| PstI
+
-
! rowspan="1"| BV
+
-
! rowspan="1"| 2100 pb
+
-
! rowspan="1"| 883 pb
+
-
|align=center|
+
-
|align=center|
+
-
|-
+
-
! rowspan="5" style="background: #ccccff;" | D120
+
-
! rowspan="5" style="background: #ccccff;" | B0030
+
-
|align=center|1
+
-
! rowspan="2"| XbaI
+
-
! rowspan="2"| PstI
+
-
! rowspan="2"| BI
+
-
! rowspan="2"| 2057 pb
+
-
! rowspan="2"| 37 pb
+
-
|align=center|
+
-
|align=center|
+
-
|-
+
-
|align=center|2
+
-
|align=center|
+
-
|align=center|
+
-
|-
+
-
|align=center|3
+
-
|align=center| EcoRI
+
-
|align=center| XbaI
+
-
|align=center| FV
+
-
|align=center| 2079 pb
+
-
|align=center| 15 pb
+
-
|align=center|
+
-
|align=center|
+
-
|-
+
-
|align=center|4
+
-
! rowspan="2"| SpeI
+
-
! rowspan="2"| PstI
+
-
! rowspan="2"| BV
+
-
! rowspan="2"| 2080 pb
+
-
! rowspan="2"| 14 pb
+
-
|align=center|
+
-
|align=center|
+
-
|-
+
-
|align=center|5
+
-
|align=center|
+
-
|align=center|
+
-
|-
+
-
! rowspan="1" style="background: #ccccff;"|D121
+
-
! rowspan="1" style="background: #ccccff;"|E0422
+
-
|1
+
-
|XbaI
+
-
|PstI
+
-
| FV
+
-
| 2057 pb
+
-
| 939 pb
+
-
|
+
-
|
+
|-
|-
-
! rowspan="1" style="background: #ccccff;"|D122
+
|align="center"| MP122
-
! rowspan="1" style="background: #ccccff;"|E0840
+
|align="center"|[http://partsregistry.org/Part:BBa_E0840 E0840]
-
|1
+
|align="center"|gfp tri-part; strong rbs
-
|XbaI
+
-
|PstI
+
-
| FV
+
-
| 2057 pb
+
-
| 900 pb
+
-
|
+
-
|
+
|}
|}

Latest revision as of 15:32, 31 July 2008

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Contents

DNA Extraction

  • Use of Promega's protocol for the extraction of: gel n°1 --> 4 : (D100 --> D110 cl-1 ES))
  • Use of Qiagen's protocol for the extraction of: gel n°5 --> 8 : (D110 cl-2 ES --> D120))
  • Analysis of the extraction success by electrophoresis on 2µl of the parts extracted.


Determination of DNA concentration by spectrophotometry

Conditions:


  • 2µl of the DNA extracted / 98µl of pure water
  • Reading against the adaptated blank (2µl of the elution's buffer / 98µl of pure water)


Results :


-D100 --> D110 cl-1 ES : Absorbance ~20-50µg/ml ; DO 260/280 = 1,5 ; DO 260/230 = 0,0-0.20

-D110 cl-2 ES --> D120 : Absorbance ~0-10µg/ml


Determination of DNA concentration by electrophoresis

  • to check for the samples from Qiagen's protocol, if the absence of detection of absorbance, is due to a problem of reading or a problem during the extraction, we realise an electrophoresis.


==> conclusion : we don't succeed to detect DNA by spectrophometry or electrophoresis for the samples produced by Qiagen's protocol.


==> we decide to repeat all the samples from the beginning, so we culture O/N at 37°c all the strains usefull for the experiments

Culture

in 10ml LB with adaptated antibiotics, all the Biobricks used before (MP100 --> MP122)

  • will be use to do minipreps


MiniPreps

  • Use of Promega's protocol on all the clones cultivated on the 24th.
  • Preparation of 50µl of minipreps in simple.


list of Minipreps

Name Biobrick Description
MP100 [http://partsregistry.org/Part:BBa_B0034 B0034] Strongest RBS (Efficiency = 1)
MP116 [http://partsregistry.org/Part:BBa_J23100 J23100] Strong constitutive promoter in J61002
MP120 [http://partsregistry.org/Part:BBa_B0030 B0030] Strong RBS (Efficiency = 0,6)
MP121 [http://partsregistry.org/Part:BBa_E0422 E0422] ECFP (RBS+LVA+Term)
MP122 [http://partsregistry.org/Part:BBa_E0840 E0840] gfp tri-part; strong rbs