Team:Paris/September 4

From 2008.igem.org

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(→Transformation results (rbs-TetR in mRFP-LVA-tripart-pSB1A3))
(→Measurement of the concentration of D168 purified)
 
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{{Paris/Calendar_Links|September 3|September 5}}
{{Paris/Calendar_Links|September 3|September 5}}
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{{Paris/Calendar_Links|September 3|September 5}}
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=Construction of rbs-TetR-mRFP-LVA-tripart (L173)=
 +
[[Image:Part_icon_rbs.png]][[Image:icon_coding.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
-
=Transformation results (rbs-TetR in mRFP-LVA-tripart-pSB1A3)=
+
==PCR Screening of L173 transformants==
 +
 
 +
===Transformation results (rbs-TetR in mRFP-LVA-tripart-pSB1A3)===
Ligation '''L173'''
Ligation '''L173'''
Line 18: Line 21:
|-
|-
|'''Number of colonies'''
|'''Number of colonies'''
 +
|many
 +
|1
 +
|0
 +
|2
 +
|0
 +
|-
 +
|}
 +
 +
===PCR Screening===
 +
 +
[[Image:KR000292.JPG|thumb|PCR screening]]
 +
 +
PCR screening programm
 +
*elongation tim: 2 min
 +
*primers used: O18 & O19
 +
*positive PCR control: S158 (pSB3K3)
 +
*negative PCR control: no template
 +
 +
{|border="1" style="text-align: center"
 +
|'''Well n°'''
 +
|1
 +
|2
 +
|3
 +
|4
 +
|5
 +
|6
 +
|7
 +
|-
 +
|'''Sample'''
 +
|rowspan="3"|1 kb <br> DNA ladder
 +
|rowspan="3"|positive PCR <br> control
 +
|rowspan="3"|negative PCR <br> control
 +
|rowspan="3"|negative transformation <br> control clone
 +
|L173.1
 +
|L173.2
 +
|rowspan="3"|100 bp <br> DNA ladder
 +
|-
 +
|'''Expected size'''
 +
|colspan="2"|'''1894 bp'''
 +
|-
 +
|'''Measured size'''
 +
|1,2 kb
 +
|1,2 kb
 +
|-
 +
|}
 +
 +
<br>'''Results''': the clones are not correct.
 +
 +
=Digestion=
 +
 +
{|border="1" style="text-align: center"
 +
|'''Digestion n°'''
 +
|'''DNA substrat'''
 +
|'''Digestion by'''
 +
|-
 +
|D112
 +
|MP106: S03879 (rbs-TetR) in pSB1A2
 +
|EcoRI & SpeI
 +
|-
 +
|}
 +
<br>
 +
*5 µL of DNA
 +
*3 µL of 10X buffer n°2
 +
*0,3 µL 100X BSA
 +
*1 µL of EcoRI
 +
*1 µL of SpeI
 +
*19,7 µL of water
 +
<br>
 +
Incubation 2h30 at 37°C and then 20 min at 65°C.
 +
<br>Not purified yet. Put at -20°C.
 +
 +
=Construction of pFlhB-mRFP-LVA-tripart & pFliL-ECFP-LVA-tripart=
 +
[[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:Part_icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
 +
*L183 = pFlhB - mRFP LVA+
 +
*L184 = pFliL - ECFP LVA+
 +
*L185 = pFliL - ECFP LVA-
 +
 +
 +
==Measurement of the concentration of the purified digestion==
 +
[[Team:Paris/Notebook/Protocols#Concentration_of_the_Miniprep | Protocol (it's same that for Miniprep)]]
 +
 +
{|border="1" style="text-align: center"
 +
|'''Digestion Name'''
 +
|'''Concentration (µg/mL)'''
 +
|'''Ratio 260/280'''
 +
|-
 +
|D134.2
 +
|114
 +
|nd
 +
|-
 +
|D134.2
 +
|84,4
 +
|nd
 +
|-
 +
|D149.2
 +
|140
 +
|nd
 +
|}
 +
 +
=>the measurement of the absorbance was so instable, that we have prefered to do it again, <br>we do it by an another thechnik : Analysis on gel.
 +
 +
==Ligation==
 +
[[Team:Paris/Notebook/Protocols#Ligation |Protocol]]
 +
 +
{|border="1" style="text-align: center"
 +
|'''Ligation Name'''
 +
|'''Vector Name'''
 +
|'''Volume Vector (µL)'''
 +
|'''Insert'''
 +
|'''Volume Insert (µL)'''
 +
|-
 +
|L183
 +
|D187
|
|
 +
|D134
|
|
 +
|-
 +
|L184
 +
|D198
|
|
-
|
+
|D149
|
|
|-
|-
 +
|L185
 +
|D199
 +
|
 +
|D149
 +
|
|}
|}
-
 
+
=>it has been missed to do adaptated control.
-
L174 = d134 + d187 (pFlhB-mRFP LVA+)
+
-
L175 = d149 + d° (pFliL - ECFP LVA+)
+
-
L176 = d149 + d° (pFliL - ECFP LVA-)
+

Latest revision as of 19:15, 16 September 2008

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Contents

Construction of rbs-TetR-mRFP-LVA-tripart (L173)

Part icon rbs.pngIcon coding.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png

PCR Screening of L173 transformants

Transformation results (rbs-TetR in mRFP-LVA-tripart-pSB1A3)

Ligation L173

  • insert: D112 (ES) rbs-TetR
  • vector: D187 (EX) mRFP-LVA-tripart-pSB1A3
Sample positive control
pUC19 		negative control
no DNA 		ligation control
(without insert) 		L173 (3:1 ratio) L173 (4:1 ratio)
Number of colonies many 1 0 2 0

PCR Screening

PCR screening

PCR screening programm

  • elongation tim: 2 min
  • primers used: O18 & O19
  • positive PCR control: S158 (pSB3K3)
  • negative PCR control: no template
Well n° 1 2 3 4 5 6 7
Sample 1 kb
DNA ladder 		positive PCR
control 		negative PCR
control 		negative transformation
control clone 		L173.1 L173.2 100 bp
DNA ladder
Expected size 1894 bp
Measured size 1,2 kb 1,2 kb


Results: the clones are not correct.

Digestion

Digestion n° DNA substrat Digestion by
D112 MP106: S03879 (rbs-TetR) in pSB1A2 EcoRI & SpeI


  • 5 µL of DNA
  • 3 µL of 10X buffer n°2
  • 0,3 µL 100X BSA
  • 1 µL of EcoRI
  • 1 µL of SpeI
  • 19,7 µL of water


Incubation 2h30 at 37°C and then 20 min at 65°C.
Not purified yet. Put at -20°C.

Construction of pFlhB-mRFP-LVA-tripart & pFliL-ECFP-LVA-tripart

Part icon regulatory.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png

  • L183 = pFlhB - mRFP LVA+
  • L184 = pFliL - ECFP LVA+
  • L185 = pFliL - ECFP LVA-


Measurement of the concentration of the purified digestion

Protocol (it's same that for Miniprep)

Digestion Name Concentration (µg/mL) Ratio 260/280
D134.2 114 nd
D134.2 84,4 nd
D149.2 140 nd 
=>the measurement of the absorbance was so instable, that we have prefered to do it again, 
we do it by an another thechnik : Analysis on gel.

Ligation

Protocol

Ligation Name Vector Name Volume Vector (µL) Insert Volume Insert (µL)
L183 D187 D134
L184 D198 D149
L185 D199 D149 
=>it has been missed to do adaptated control.
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