Team:Paris/August 28

From 2008.igem.org

(Difference between revisions)
(→Transformation of OmpR*)
(→Repeat of Digestion)
 
(12 intermediate revisions not shown)
Line 2: Line 2:
='''Transformation of OmpR*'''=
='''Transformation of OmpR*'''=
-
='''Construction of pFlhB - mRFP Tripart (LVA+)'''=
 
-
Aim : Construction of ''' "pFlhB-RBS-mRFP-dbl ter" (pFlhB-I732078)''' [[Image:Part_icon_regulatory.png]][[Image:Part_icon_rbs.png]][[Image:icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]]
+
 
-
We can only do the construction with mRFP Tripart (LVA+) because the stable strain with the Biobricks I732011 (mRFP Tripart LVA-) don't to growth.
+
=Cloning of EnvZ*=
 +
 
 +
The cloning of EnvZ* didn't worked. So we decided to try one more time, starting from the PCR product.
==Digestion==
==Digestion==
-
==='''Measurement of the concentration of D187 purified'''===
 
-
[[Team:Paris/Notebook/Protocols#Concentration_of_the_Miniprep | Protocol (it's same that for Miniprep)]]
+
[[Image:EnvZ pSB1A2.JPG|thumb|Digestion of EnvZ* and lasR-pSB1A2]]
{|border="1" style="text-align: center"
{|border="1" style="text-align: center"
-
|'''Digestion Name'''
+
|'''Digestion n°'''
-
|'''Concentration (µg/mL)'''
+
|'''Name'''
-
|'''Ratio 260/280'''
+
|'''volume of DNA digested'''
 +
|'''total volume reaction'''
 +
|'''enzymes used'''
|-
|-
-
|D187
+
|D159
-
|
+
|PCR147 (EnvZ*)
-
|
+
|3 µL
 +
|30 µL
 +
|XbaI & PstI
 +
|-
 +
|D116
 +
|MP108 (lasR-pSB1A2)
 +
|2 µL
 +
|30 µL
 +
|XbaI & PstI
|}
|}
 +
<br>
 +
'''Purification'''
 +
*EnvZ*: directly by the Qiagen kit
 +
*pSB1A2: excision of the band of interest (2 kb) and then purification using the Qiagen kit
-
==Ligation==
+
=Freezer empty=
-
[[Team:Paris/Notebook/Protocols#Ligation |Protocol]]
+
[[Team:Paris/Notebook/Protocols#Digestion |Protocol]]
-
{|border="1" style="text-align: center"
+
We did again [[Team:Paris/Notebook/Freezer#Digestion Products |D110]] and  [[Team:Paris/Notebook/Freezer#Digestion Products |D125]].
-
|'''Ligation Name'''
+
-
|'''Vector Name'''
+
-
|'''Volume Vector (µL)'''
+
-
|'''Insert'''
+
-
|'''Volume Insert (µL)'''
+
-
|-
+
-
|L168
+
-
|D187
+
-
|
+
-
|D186
+
-
|
+
-
|-
+
-
|Control L168
+
-
|D166
+
-
|
+
-
| -
+
-
|}
+

Latest revision as of 19:09, 9 September 2008

← Yesterday

↓ Calendar ↑

Tomorrow →

Contents

Transformation of OmpR*

Cloning of EnvZ*

The cloning of EnvZ* didn't worked. So we decided to try one more time, starting from the PCR product.

Digestion

Digestion of EnvZ* and lasR-pSB1A2
Digestion n° Name volume of DNA digested total volume reaction enzymes used
D159 PCR147 (EnvZ*) 3 µL 30 µL XbaI & PstI
D116 MP108 (lasR-pSB1A2) 2 µL 30 µL XbaI & PstI


Purification

  • EnvZ*: directly by the Qiagen kit
  • pSB1A2: excision of the band of interest (2 kb) and then purification using the Qiagen kit

Freezer empty

Protocol

We did again D110 and D125.

Retrieved from "http://2008.igem.org/Team:Paris/August_28"