Team:Paris Saclay/Notebook/July/8

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==''Summary:''==
==''Summary:''==
FNR regulator system:
FNR regulator system:
-
*performed a digestion for AmilCP and LacZ with Not I, Xho I, EcoR I and PST I.
+
*performed another digestion for AmilCP plus sequence LacZ with Not I, Xho I, EcoR I and PST I.
*electrophoresis for those digest products
*electrophoresis for those digest products
*enrichment culture for clone of the briobrick BBa_K1155000  
*enrichment culture for clone of the briobrick BBa_K1155000  
Line 18: Line 18:
<p>For some of the enzymes which are possible to use one common buffer, we did double digest
<p>For some of the enzymes which are possible to use one common buffer, we did double digest
 +
{| border="1" align="center"
 +
|-
 +
|DNA
 +
|enzyme
 +
|buffer
 +
|-
 +
|AmilCP1
 +
|Not I
 +
|Orange
 +
|-
 +
|AmilCP2
 +
|Not I
 +
|Orange
 +
|-
 +
|AmilCP1
 +
|Xho I
 +
|Red
 +
|-
 +
|AmilCP2
 +
|Xho I
 +
|Red
 +
|-
 +
|AmilCP1
 +
|EcoR I
 +
|Orange
 +
|-
 +
|AmilCP2
 +
|EcoR I
 +
|Orange
 +
|-
 +
|LacZ1
 +
|EcoR I + PST I
 +
|Orange
 +
|-
 +
|LacZ2
 +
|EcoR I + PST I
 +
|Orange
 +
|}
 +
<br>
 +
<div>Volume added:</div>
 +
{| border="1" align="center"
 +
|-
 +
|DNA
 +
|2(µl)
 +
|-
 +
|buffer
 +
|2(µl)
 +
|-
 +
|Enzyme(foreach)
 +
|0.5(µl)
 +
|-
 +
|H20
 +
|15.5(µl)
 +
|-
 +
|total
 +
|20(µl)
 +
|}
-
DNA enzyme buffer
+
<br>
-
AmilCP1 Not I Orange
+
-
AmilCP2 Not I Orange
+
-
AmilCP1 Xho I Red
+
-
AmilCP2 Xho I Red
+
-
AmilCP1 EcoR I Orange
+
-
AmilCP2 EcoR I Orange
+
-
LacZ1 EcoR I + PST I Orange
+
-
LacZ2 EcoR I + PST I Orange
+
-
Volume added:
+
The electrophoresis (135 V for 30 minutes) for digest products were migrated in agarose gel (0.5X).
-
DNA 2(µl)
+
-
buffer 2(µl)
+
-
Enzyme(foreach) 0.5(µl)
+
-
H20 15.5(µl)
+
-
total 20(µl)
+
-
Notes: by using double digest, an informatics tool available in Fermentas web site, it is possible to use one buffer for both EcoR I and PST I.
+
Results:
-
2. The electrophoresis (135 V for 30 minutes) for digest products were migrated in agarose gel (0.5X).
+
-
Volume added: for each well
 
-
plasmid 10µl
 
-
OR Pfnr 5µl
 
-
Blue
 
-
Results:
 
   
   
-
3. Overnight incubation at 37°C with agitation.
+
Overnight incubation at 37°C with agitation.
-
4. The electrophoresis for the plasmids which we had extracted last week
+
The electrophoresis for the plasmids which we had extracted last week
Volume added:  
Volume added:  

Revision as of 09:34, 21 September 2013

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Notebook : July 8

Summary:

FNR regulator system:

  • performed another digestion for AmilCP plus sequence LacZ with Not I, Xho I, EcoR I and PST I.
  • electrophoresis for those digest products
  • enrichment culture for clone of the briobrick BBa_K1155000
  • electrophoresis for extracted products of last friday


Lab work

Restriction digest

For some of the enzymes which are possible to use one common buffer, we did double digest

DNA enzyme buffer
AmilCP1 Not I Orange
AmilCP2 Not I Orange
AmilCP1 Xho I Red
AmilCP2 Xho I Red
AmilCP1 EcoR I Orange
AmilCP2 EcoR I Orange
LacZ1 EcoR I + PST I Orange
LacZ2 EcoR I + PST I Orange


Volume added:
DNA 2(µl)
buffer 2(µl)
Enzyme(foreach) 0.5(µl)
H20 15.5(µl)
total 20(µl)


The electrophoresis (135 V for 30 minutes) for digest products were migrated in agarose gel (0.5X).

Results:




Overnight incubation at 37°C with agitation. The electrophoresis for the plasmids which we had extracted last week

Volume added: Pfnr2 R1P R2 A1P 10µl 10µl 10µl 10µl

Migration at 100V for 25mins then continued till 45 minutes: Results:


The concentration of Pfnr2 was detected by NanoDrop : C = 1788.8 ng/µl 260/280 = 2.08 We considered that our extraction of plasmid DNA was not successful, we need to perform another extraction DNA.



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