Team:Paris/August 25

From 2008.igem.org

(Difference between revisions)
(→Results of the transformation we did the day before yesterday)
Line 79: Line 79:
='''Cloning of EnvZ*'''=
='''Cloning of EnvZ*'''=
 +
The sequencing of EnvZ* previously cloned, revealed a loss of about 300 bp. EnvZ* contains indeed an EcoRI restriction site within its sequence. So we can't use this enzyme during the cloning.
 +
 +
==Digestion==
 +
 +
{|border="1" style="text-align: center"
 +
|'''name'''
 +
|'''sample'''
 +
|'''digestion'''
 +
|'''digestion number'''
 +
|-
 +
|EnvZ*
 +
|PCR129 from August 8th
 +
|XbaI & PstI
 +
|D159
 +
|-
 +
|pSB1A2
 +
|MP108 (C0179 (lasR-pSB1A2))
 +
|XbaI & PstI
 +
|D116
 +
|-
 +
|}
 +
 +
'''Reaction mixture'''
 +
*4 µL of PCR129 (or 2 µL of MP108)
 +
*3 µL of 10X buffer n°2
 +
*0,3 µL of 100X BSA
 +
*1 µL of XbaI
 +
*1 µL of PstI
 +
*20,7 µL (or 22,7 µL) of water
 +
Incubation at 37°C during 2H25, and then ~20 min at 65°C
 +
 +
==Electrophoresis==
 +
 +
1% agarose gel
 +
*EnvZ*: 3 µL of digestion products + 1 µL of loading blue + 2 µL of water
 +
*pSB1A2: 30 µL of digestion products + 6 µL of loading blue
 +
[[Image:KR000225.jpg|thumb|]]
[[Image:KR000225.jpg|thumb|]]
[[Image:KR000226.jpg|thumb|Gel after excision]]
[[Image:KR000226.jpg|thumb|Gel after excision]]
 +
 +
{|border="1" style="text-align: center"
 +
|'''well n°'''
 +
|1
 +
|2
 +
|3
 +
|4
 +
|5
 +
|6
 +
|-
 +
|'''sample'''
 +
|1 kb DNA ladder
 +
|D159 (EnvZ*)
 +
|
 +
|D116 (pSB1A2 & lasR)
 +
|
 +
|100 bp DNA ladder
 +
|-
 +
|'''expected size'''
 +
|
 +
|1421 bp
 +
|
 +
|2057 bp & 707 bp
 +
|
 +
|
 +
|-
 +
|'''measured size'''
 +
|
 +
|1,4 kb
 +
|
 +
|2 kb & 0.7 kb
 +
|
 +
|
 +
|-
 +
|}

Revision as of 09:56, 26 August 2008

← Yesterday

↓ Calendar ↑

Tomorrow →

Contents

Construction for Synchronization

Transformation of the ligations we did yesterday

Ligation Name Description Antibio
L159 D125 (FV) + D109 (FI) Kan
Control L159 D125 Kan
L162 D107 (BV) + D163 (BI) Amp
Control L162 D107 Amp
L163 D110 (BV) + D163 (BI) Amp
Control L163 D110 Amp


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


Results of the transformation we did the day before yesterday

Ligation name Description Antibio Number Colonies observed Fluorescence Comments
Ligations
L164 D168(FV) - D132(FI)
pFlgA - gfp generator 		Amp 125 No ok
Controls
TL164 D168(FV) Amp 8 No ok
Positive Control pUC19 Amp 2000 (efficiency 2.10^8) No OK 
=> Need to screen to know which clones we can use for the  of  pFlgA promotor characterization.

Cloning of EnvZ*

The sequencing of EnvZ* previously cloned, revealed a loss of about 300 bp. EnvZ* contains indeed an EcoRI restriction site within its sequence. So we can't use this enzyme during the cloning.

Digestion

name sample digestion digestion number
EnvZ* PCR129 from August 8th XbaI & PstI D159
pSB1A2 MP108 (C0179 (lasR-pSB1A2)) XbaI & PstI D116

Reaction mixture

  • 4 µL of PCR129 (or 2 µL of MP108)
  • 3 µL of 10X buffer n°2
  • 0,3 µL of 100X BSA
  • 1 µL of XbaI
  • 1 µL of PstI
  • 20,7 µL (or 22,7 µL) of water

Incubation at 37°C during 2H25, and then ~20 min at 65°C

Electrophoresis

1% agarose gel

  • EnvZ*: 3 µL of digestion products + 1 µL of loading blue + 2 µL of water
  • pSB1A2: 30 µL of digestion products + 6 µL of loading blue
KR000225.jpg
Gel after excision
well n° 1 2 3 4 5 6
sample 1 kb DNA ladder D159 (EnvZ*) D116 (pSB1A2 & lasR) 100 bp DNA ladder
expected size 1421 bp 2057 bp & 707 bp
measured size 1,4 kb 2 kb & 0.7 kb
Retrieved from "http://2008.igem.org/Team:Paris/August_25"