Team:Paris/August 22

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=Analysis of the transformant of FlhDC+promotor=
=Analysis of the transformant of FlhDC+promotor=
-
='''transformation of the ligations we did yesterday'''=
+
*Analysis of the plasmids MP160.1 and MP160.2 (FlhDC+promotor in pSB1A2)
 +
*Control: MP143 (GFP generator in pSB1A2)
 +
==PCR==
 +
PCR screening programm
 +
<br>elongation time: 1 min 30
 +
<br>total volume reaction (25 µL)
 +
*12,5 µL Quick load PCR Mix 2X
 +
*0,5 µL O18
 +
*0,5 µL O19
 +
*1 µL DNA
 +
*10,5 µL water
 +
==Digestion==
 +
total volume reaction (30 µL)
 +
*2 µL DNA
 +
*3 µL buffer 2 10X
 +
*0,3 µL BSA 100X
 +
*1 µL XbaI
 +
*1 µL SpeI
 +
*22,7 µL water
 +
Incubation 2h55 at 37°C and then 20 min at 65°C.
 +
 +
==Electrophoresis==
 +
 +
[[Image:KR000219.jpg|thumb|]]
 +
 +
*1% agarose gel
 +
*for PCR products: 10 µL loaded
 +
*for digestion products (30 µL): adding of 7 µL of loading blue and then 20 µL loaded on gel
 +
 +
{|border="1" style="text-align: center"
 +
|'''well n°'''
 +
|1
 +
|2
 +
|3
 +
|4
 +
|5
 +
|6
 +
|7
 +
|8
 +
|9
 +
|-
 +
|'''method'''
 +
|
 +
|colspan="4"|'''PCR'''
 +
|colspan="3"|'''digestion'''
 +
|
 +
|-
 +
|'''sample'''
 +
|1 kb DNA ladder
 +
|positive control MP143
 +
|negative control
 +
|MP160.1
 +
|MP160.2
 +
|MP143
 +
|MP160.1
 +
|MP160.2
 +
|100 bp DNA ladder
 +
|-
 +
|'''expected size'''
 +
|
 +
|'''1114 bp'''
 +
|
 +
|colspan="2"|'''1403 bp'''
 +
|'''876 bp''' (E0240)<br>'''2079''' (pSB1A2)
 +
|colspan="2"|'''1165 bp''' (FlhDC+promotor)<br>'''2079 bp''' (pSB1A2)
 +
|
 +
|-
 +
|'''measured size'''
 +
|
 +
|style="background: #ff6d73" |'''1 kb'''<br>3 kb
 +
|
 +
|style="background: #ff6d73" |0,3 kb<br>1,8 kb
 +
|style="background: #cbff7B"| 0,3 kb<br>1,8 kb
 +
|style="background: #ff6d73" |0,9 kb<br>2 kb
 +
|style="background: #ff6d73" |2 kb
 +
|style="background: #ff6d73" |2 kb
 +
|
 +
|}
 +
 +
 +
'''Results''': The clones tested didn't have the insert.
 +
 +
 +
='''Construction of pFlgA - GFP Generator'''=
 +
 +
Aim : Construction of ''' "pFlgA-RBS-GFP-dbl ter" (pFlgA-E0240)''' [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
 +
==Digestion==
 +
===Digestion===
 +
[[Team:Paris/Notebook/Protocols#Digestion|Protocol Digestion]]
 +
 +
{| border="1" style="text-align: center"
 +
|'''Name'''
 +
|'''Template DNA'''
 +
|'''Description'''
 +
|'''Vol MP (µl)'''
 +
|'''Vol H2O (µl)'''
 +
|'''Enzymes'''
 +
|-
 +
|D168
 +
|MP143.1
 +
| RBS - GFP - term  (FV)
 +
|7.25
 +
|17.45
 +
|EcoRI and XbaI
 +
|}
 +
 +
==='''Gel Extraction'''===
 +
[[Team:Paris/Notebook/Protocols#Migration after extraction |Protocol]]
 +
[[Image:KR000222b.JPG |thumb |Gel Extraction of D168]]
 +
 +
{|border="1" style="text-align: center"
 +
|'''Well'''
 +
|1
 +
|2
 +
|3
 +
|4
 +
|5
 +
|6
 +
|-
 +
|'''Sample'''
 +
|100 pb ladder
 +
|MP143.1
 +
|no sample
 +
|D168
 +
|colspan="2"|no sample
 +
|-
 +
|'''Expected size (pb)'''
 +
|
 +
|
 +
|
 +
|2 940
 +
| colspan="2"|
 +
|-
 +
|'''Measured size (pb)'''
 +
|
 +
|style="background: #cbff7B"|5.000
 +
|
 +
|style="background: #cbff7B"|3 000
 +
|colspan="2"|
 +
|}
 +
 +
==='''Measurement of the concentration of D168 purified'''===
 +
 +
[[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'''
 +
|-
 +
|D168
 +
|24
 +
|2.15
 +
|}
 +
 +
==Ligation==
 +
[[Team:Paris/Notebook/Protocols#Ligation |Protocol]]
 +
 +
{|border="1" style="text-align: center"
 +
|'''Ligation Name'''
 +
|'''Vector Name'''
 +
|'''Volume Vector (µL)'''
 +
|'''Insert'''
 +
|'''Volume Insert (µL)'''
 +
|-
 +
|L164
 +
|D168
 +
|1.67
 +
|D132
 +
|2.04
 +
|-
 +
|Control L164
 +
|D168
 +
|1.67
 +
| -
 +
| -
 +
|}
 +
 +
 +
='''Construction for FIFO'''=
 +
 +
Aim : Construction of pFlgA - YFP tripart (+/- LVA)''' "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432)''' [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
 +
 +
=='''Transformation of the ligations we did [[Team:Paris/August 21 |yesterday]]'''==
 +
[[Team:Paris/Notebook/Protocols#Transformation |Protocol]]
 +
 +
{|border="1" style="text-align:center"
 +
|'''Ligation Name'''
 +
|'''Description'''
 +
|'''Antibio'''
 +
|-
 +
|L160
 +
|D166 (FV) + D132 (FI)<br>pFlgA - YFP  tripart (LVA-)
 +
|Amp
 +
|-
 +
|TL160
 +
|D166<br> YFP  tripart (LVA-)
 +
|Amp
 +
|-
 +
|L161
 +
|D167 (FV) + D132 (FI)<br>pFlgA - YFP  tripart (LVA+)
 +
|Amp
 +
|-
 +
|TL161
 +
|D167<br>YFP  tripart (LVA+)
 +
|Amp
 +
|}
='''Construction for synchronization'''=
='''Construction for synchronization'''=
-
=='''Ligations'''==
+
=='''Transformation of the ligations we did [[Team:Paris/August 21 |yesterday]]'''==
 +
[[Team:Paris/Notebook/Protocols#Transformation |Protocol]]
 +
 
 +
{|border="1" style="text-align:center"
 +
|'''Ligation Name'''
 +
|'''Description'''
 +
|'''Antibio'''
 +
|-
 +
|L158
 +
|D110 (BV) + D131 (BI)
 +
|Amp
 +
|-
 +
|Control L158
 +
|D110
 +
|Amp
 +
|-
 +
|L159
 +
|D125 (FV) + D109 (FI)
 +
|Kan
 +
|-
 +
|Control L159
 +
|D125
 +
|Kan
 +
|}
 +
 
 +
=='''Ligation'''==
[[Team:Paris/Notebook/Protocols#Ligation |Protocol]]
[[Team:Paris/Notebook/Protocols#Ligation |Protocol]]
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|'''Volume vector (µL)'''
|'''Volume vector (µL)'''
|'''Insert Name'''
|'''Insert Name'''
-
|'''Volume insert(µL)'''
+
|'''Volume insert (µL)'''
|-
|-
|L162
|L162
Line 24: Line 256:
|D163 (BI)
|D163 (BI)
|3.46
|3.46
 +
|-
 +
|Control L162
 +
|autoligation control
 +
|D107 (BV)
 +
|10
 +
| -
 +
| -
|-
|-
|L163
|L163
Line 31: Line 270:
|D163 (BI)
|D163 (BI)
|3.44
|3.44
 +
|-
 +
|Control L163
 +
|autoligation control
 +
|D110 (BV)
 +
|2.5
 +
| -
 +
| -
 +
|}
 +
 +
=='''Transformation'''==
 +
[[Team:Paris/Notebook/Protocols#transformation |Protocol]]
 +
 +
{|border="1" style="text-align:center"
 +
|'''Ligation Name'''
 +
|'''Description'''
 +
|'''Antibio'''
 +
|-
 +
|L162
 +
|D107 (BV) + D163 (BI)
 +
|Amp
 +
|-
 +
|Control L162
 +
|D107
 +
|Amp
 +
|-
 +
|L163
 +
|D110 (BV) + D163 (BI)
 +
|Amp
 +
|-
 +
|Control L163
 +
|D110
 +
|Amp
 +
|}
 +
 +
 +
 +
='''Promoter characterization plasmids'''=
 +
 +
==Transformation of ligations from August 20th==
 +
[[Team:Paris/Notebook/Protocols#Transformation |Protocol]]
 +
 +
'''Top 10 cells were used'''
 +
 +
{|border="1" style="text-align: center"
 +
|'''Ligation name'''
 +
|'''Vector digestion'''
 +
|'''Vector description'''
 +
|'''C° Vector (µg/mL)'''
 +
|'''V. Vector (µl)'''
 +
|'''Insert digestion'''
 +
|'''Insert description'''
 +
|'''C° Insert (µg/mL)'''
 +
|'''V. Insert (µl)'''
 +
|'''Product description'''
 +
|'''Antibiotic'''
 +
|-
 +
|L155
 +
|D164
 +
|J23101 promoter
 +
|3
 +
|16
 +
|D163
 +
|gfp generator
 +
|14
 +
|8
 +
|J23101 promoter-gfp generator
 +
|Amp
 +
|-
 +
|L156
 +
|D161
 +
|pTet promoter
 +
|39
 +
|1.25
 +
|D163
 +
|gfp generator
 +
|14
 +
|5
 +
|pTet promoter-gfp generator
 +
|Kana
 +
|-
 +
|Control L156
 +
|D161
 +
|
 +
|
 +
|1.25
 +
|none
 +
|
 +
|
 +
|
 +
|Vector autoligation control
 +
|Kana
 +
|-
 +
|L157
 +
|D125.2
 +
|B0015
 +
|15
 +
|3
 +
|D162
 +
|tetR
 +
|11
 +
|4
 +
|tetR-B0015
 +
|Kana
 +
|-
 +
|Control L157
 +
|D125.2
 +
|
 +
|
 +
|3
 +
|none
 +
|
 +
|
 +
|
 +
|Vector autoligation control
 +
|Kana
 +
|-
 +
|L166
 +
|D185
 +
|RBS B0032
 +
|21
 +
|2
 +
|D182
 +
|tetR
 +
|10
 +
|6.5
 +
|RBS B0032 - tetR
 +
|Amp
 +
|-
 +
|Control L166
 +
|D185
 +
|
 +
|
 +
|2
 +
|none
 +
|
 +
|
 +
|
 +
|Vector autoligation control
 +
|Amp
 +
|-
 +
|L167
 +
|D181
 +
|pTet
 +
|1
 +
|14
 +
|D184
 +
|gfp generator
 +
|14
 +
|3
 +
|gfp generator - pTet
 +
|Amp
 +
|-
 +
|Control L167
 +
|D181
 +
|pTet
 +
|
 +
|14
 +
|none
 +
|
 +
|
 +
|
 +
|Vector autoligation control
 +
|Amp
|}
|}

Latest revision as of 00:54, 21 September 2008

← Yesterday

↓ Calendar ↑

Tomorrow →

Contents

Analysis of the transformant of FlhDC+promotor

  • Analysis of the plasmids MP160.1 and MP160.2 (FlhDC+promotor in pSB1A2)
  • Control: MP143 (GFP generator in pSB1A2)

PCR

PCR screening programm
elongation time: 1 min 30
total volume reaction (25 µL)

  • 12,5 µL Quick load PCR Mix 2X
  • 0,5 µL O18
  • 0,5 µL O19
  • 1 µL DNA
  • 10,5 µL water

Digestion

total volume reaction (30 µL)

  • 2 µL DNA
  • 3 µL buffer 2 10X
  • 0,3 µL BSA 100X
  • 1 µL XbaI
  • 1 µL SpeI
  • 22,7 µL water

Incubation 2h55 at 37°C and then 20 min at 65°C.

Electrophoresis

KR000219.jpg
  • 1% agarose gel
  • for PCR products: 10 µL loaded
  • for digestion products (30 µL): adding of 7 µL of loading blue and then 20 µL loaded on gel
well n° 1 2 3 4 5 6 7 8 9
method PCR digestion
sample 1 kb DNA ladder positive control MP143 negative control MP160.1 MP160.2 MP143 MP160.1 MP160.2 100 bp DNA ladder
expected size 1114 bp 1403 bp 876 bp (E0240)
2079 (pSB1A2) 		1165 bp (FlhDC+promotor)
2079 bp (pSB1A2)
measured size 1 kb
3 kb 		0,3 kb
1,8 kb 		0,3 kb
1,8 kb 		0,9 kb
2 kb 		2 kb 2 kb 


Results: The clones tested didn't have the insert.


Construction of pFlgA - GFP Generator

Aim : Construction of "pFlgA-RBS-GFP-dbl ter" (pFlgA-E0240) Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png

Digestion

Digestion

Protocol Digestion

Name Template DNA Description Vol MP (µl) Vol H2O (µl) Enzymes
D168 MP143.1 RBS - GFP - term (FV) 7.25 17.45 EcoRI and XbaI

Gel Extraction

Protocol

Gel Extraction of D168
Well 1 2 3 4 5 6
Sample 100 pb ladder MP143.1 no sample D168 no sample
Expected size (pb) 2 940
Measured size (pb) 5.000 3 000

Measurement of the concentration of D168 purified

Protocol (it's same that for Miniprep)

Digestion Name Concentration (µg/mL) Ratio 260/280
D168 24 2.15

Ligation

Protocol

Ligation Name Vector Name Volume Vector (µL) Insert Volume Insert (µL)
L164 D168 1.67 D132 2.04
Control L164 D168 1.67 - -


Construction for FIFO

Aim : Construction of pFlgA - YFP tripart (+/- LVA) "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432) Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png

Transformation of the ligations we did yesterday

Protocol

Ligation Name Description Antibio
L160 D166 (FV) + D132 (FI)
pFlgA - YFP tripart (LVA-) 		Amp
TL160 D166
YFP tripart (LVA-) 		Amp
L161 D167 (FV) + D132 (FI)
pFlgA - YFP tripart (LVA+) 		Amp
TL161 D167
YFP tripart (LVA+) 		Amp

Construction for synchronization

Transformation of the ligations we did yesterday

Protocol

Ligation Name Description Antibio
L158 D110 (BV) + D131 (BI) Amp
Control L158 D110 Amp
L159 D125 (FV) + D109 (FI) Kan
Control L159 D125 Kan

Ligation

Protocol

Ligation Name Description Vector Name Volume vector (µL) Insert Name Volume insert (µL)
L162 rbs-lasI + gfp generator
Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png 		D107 (BV) 10 D163 (BI) 3.46
Control L162 autoligation control D107 (BV) 10 - -
L163 rbs-TetR + gfp generator
Part icon rbs.pngIcon coding.pngPart icon rbs.pngPart icon reporter.pngPart icon terminator.pngPart icon terminator.png 		D110 (BV) 2.5 D163 (BI) 3.44
Control L163 autoligation control D110 (BV) 2.5 - -

Transformation

Protocol

Ligation Name Description Antibio
L162 D107 (BV) + D163 (BI) Amp
Control L162 D107 Amp
L163 D110 (BV) + D163 (BI) Amp
Control L163 D110 Amp


Promoter characterization plasmids

Transformation of ligations from August 20th

Protocol

Top 10 cells were used

Ligation name Vector digestion Vector description C° Vector (µg/mL) V. Vector (µl) Insert digestion Insert description C° Insert (µg/mL) V. Insert (µl) Product description Antibiotic
L155 D164 J23101 promoter 3 16 D163 gfp generator 14 8 J23101 promoter-gfp generator Amp
L156 D161 pTet promoter 39 1.25 D163 gfp generator 14 5 pTet promoter-gfp generator Kana
Control L156 D161 1.25 none Vector autoligation control Kana
L157 D125.2 B0015 15 3 D162 tetR 11 4 tetR-B0015 Kana
Control L157 D125.2 3 none Vector autoligation control Kana
L166 D185 RBS B0032 21 2 D182 tetR 10 6.5 RBS B0032 - tetR Amp
Control L166 D185 2 none Vector autoligation control Amp
L167 D181 pTet 1 14 D184 gfp generator 14 3 gfp generator - pTet Amp
Control L167 D181 pTet 14 none Vector autoligation control Amp
Retrieved from "http://2008.igem.org/Team:Paris/August_22"