Team:Paris Saclay/Notebook/July/8

From 2013.igem.org

(Difference between revisions)
(→Lab work)
(→2 - Electrophoresis of the digestion of BBa_K592009, BBa_I732017 by NotI, XhoI, EcoRI, EcoRI/PstI)
 
(13 intermediate revisions not shown)
Line 1: Line 1:
{{Team:Paris_Saclay/incl_debut_generique}}
{{Team:Paris_Saclay/incl_debut_generique}}
 +
='''Notebook : July 8'''=
='''Notebook : July 8'''=
-
 
-
 
-
 
-
==''Summary:''==
 
-
FNR regulator system:
 
-
*performed another digestion for AmilCP, Pfnr 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'''==
=='''Lab work'''==
-
<u>Restriction digest</u>
+
==='''A - Aerobic/Anaerobic regulation system'''===
-
<br>
+
-
<p>For some of the enzymes which are possible to use one common buffer, we did double digest
+
===='''Objective : BBa_K1155003, BBa_K1155007'''====
-
{| 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)
+
-
|}
+
-
<br>
+
===='''1 - Digestion of BBa_K592009, BBa_I732017 by NotI, XhoI, EcoRI, EcoRI/PstI'''====
-
The electrophoresis (135 V for 30 minutes) for digest products were migrated in agarose gel (0.5X).
+
Abou, Anaïs, Sheng, Zhou
-
Results:
+
Used quantities :  
-
{| align="center"
+
* NotI :  
-
| style="width:350px;border:1px solid black;" | [[File:PS080713dig.jpg|center|350px]]
+
** BBa_K592009 : 2µL
-
| style="width:350px;border:1px solid black;" |
+
** Buffer orange : 2µL
-
*Well 1,12 : marker
+
** NotI : 0.5µL
-
*Well 2: AmilCP1 + Ecor I
+
** H2O : 15.5µL
-
*Well 3: AmilCP2 + Ecor I
+
-
*Well 4: AmilCp1 + Not I
+
-
*Well 5: AmilCP2 + Not I
+
-
*Well 6: AmilCp1 + Xho I
+
-
*Well 7: AmilCp1 + Xho I
+
-
*Well 8: LacZ 1 + Ecor I + Pst I
+
-
*Well 9: LacZ 2 + Ecor I + Pst I
+
-
*Well 10: Nothing
+
-
*Well 11: fnr promoter
+
-
*gel 0.8%
+
-
|}<br>
+
 +
* XhoI :
 +
** BBa_K592009 : 2µL
 +
** Buffer red : 2µL
 +
** XhoI : 0.5µL
 +
** H2O : 15.5µL
 +
* EcoRI :
 +
** BBa_K592009 : 2µL
 +
** Buffer orange : 2µL
 +
** EcoRI : 0.5µL
 +
** H2O : 15.5µL
-
Overnight incubation at 37°C with agitation.
+
* EcoRI/PstI :
-
<br>
+
** BBa_I732017 : 2µL
-
The electrophoresis for the plasmids which we had extracted last week plus 3 other BioBrick promoter Bph R1, promoter Bph A1, Bph R2.
+
** Buffer orange : 2µL
 +
** EcoRI : 0.5µL
 +
** PstI : 0.5µL
 +
** H2O : 15µL
-
Volume added:
+
We let our digestion 1h30 at 37°C.
-
*Pfnr2: 10µl
+
-
*R1P: 10µl
+
-
*R2: 10µl
+
-
*A1P: 10µl
+
-
+
-
Migration at 100V for 25mins then continued till 45 minutes:
+
===='''2 - Electrophoresis of the digestion of BBa_K592009, BBa_I732017 by NotI, XhoI, EcoRI, EcoRI/PstI'''====
-
<br>
+
-
Results:
+
-
{| align="center"
+
Abdou, Anaïs, Sheng, Zhou
-
| style="width:350px;border:1px solid black;" | [[File:PS08071345.jpg|right|350px]]
+
-
| style="width:350px;border:1px solid black;" |
+
-
*Well 1 : R2
+
-
*Well 2 : R1P
+
-
*Well 3 : A1P
+
-
*Well 4 : Pfnr
+
-
*gel 0.8%
+
-
|}<br>
+
-
+
-
+
-
The concentration of Pfnr2 was detected by NanoDrop :
+
-
*C = 1788.8 ng/µl
+
{|
-
*260/280 = 2.08
+
| style="width:350px;border:1px solid black;" |[[File:Psgel0807.jpg|500px]]
 +
| style="width:350px;border:1px solid black;vertical-align:top;" |
 +
* Well 1 : 6µL DNA Ladder
 +
* Well 2 : 5µL BBa_K592009 clone 1 digested by EcoRI+1µL of 6X loading dye
 +
* Well 3 : 5µL BBa_K592009 clone 2 digested by EcoRI+1µL of 6X loading dye
 +
* Well 4 : 5µL BBa_K592009 clone 1 digested by NotI+1µL of 6X loading dye
 +
* Well 5 : 5µL BBa_K592009 clone 2 digested by NotI+1µL of 6X loading dye
 +
* Well 6 : 5µL BBa_K592009 clone 1 digested by XhoI+1µL of 6X loading dye
 +
* Well 7 : 5µL BBa_K592009 clone 2 digested by XhoI+1µL of 6X loading dye
 +
* Well 8 : 5µL BBa_I732017 clone 1 digested by EcoRI/PstI+1µL of 6X loading dye
 +
* Well 9 : 5µL BBa_I732017 clone 2 digested by EcoRI/PstI+1µL of 6X loading dye
 +
* Well 10 : -
 +
* Well 11 : -
 +
* Well 12 : 6µL DNA Ladder
 +
* Gel : 1%
 +
|}
-
We considered that our extraction of plasmid DNA was not successful, we need to perform another extraction DNA.
+
Expected sizes :
 +
* BBa_K592009 digested by NotI : 2046bp + 693bp
 +
* BBa_K592009 digested by XhoI : 1842bp + 892bp
 +
* BBa_I732017 : 3093bp
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
 +
We obtain fragments at the right size. Our extraction was good.
 +
|}
-
<br>
 
{| border="1" align="center"
{| border="1" align="center"
|[[Team:Paris Saclay/Notebook/July/5|<big>Previous week</big>]]
|[[Team:Paris Saclay/Notebook/July/5|<big>Previous week</big>]]
Line 143: Line 82:
|[[Team:Paris Saclay/Notebook/July/9|<big>Next day</big>]]
|[[Team:Paris Saclay/Notebook/July/9|<big>Next day</big>]]
|}
|}
-
 
-
 
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 23:55, 4 October 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : July 8

Lab work

A - Aerobic/Anaerobic regulation system

Objective : BBa_K1155003, BBa_K1155007

1 - Digestion of BBa_K592009, BBa_I732017 by NotI, XhoI, EcoRI, EcoRI/PstI

Abou, Anaïs, Sheng, Zhou

Used quantities :

  • NotI :
    • BBa_K592009 : 2µL
    • Buffer orange : 2µL
    • NotI : 0.5µL
    • H2O : 15.5µL
  • XhoI :
    • BBa_K592009 : 2µL
    • Buffer red : 2µL
    • XhoI : 0.5µL
    • H2O : 15.5µL
  • EcoRI :
    • BBa_K592009 : 2µL
    • Buffer orange : 2µL
    • EcoRI : 0.5µL
    • H2O : 15.5µL
  • EcoRI/PstI :
    • BBa_I732017 : 2µL
    • Buffer orange : 2µL
    • EcoRI : 0.5µL
    • PstI : 0.5µL
    • H2O : 15µL

We let our digestion 1h30 at 37°C.

2 - Electrophoresis of the digestion of BBa_K592009, BBa_I732017 by NotI, XhoI, EcoRI, EcoRI/PstI

Abdou, Anaïs, Sheng, Zhou

Psgel0807.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL BBa_K592009 clone 1 digested by EcoRI+1µL of 6X loading dye
  • Well 3 : 5µL BBa_K592009 clone 2 digested by EcoRI+1µL of 6X loading dye
  • Well 4 : 5µL BBa_K592009 clone 1 digested by NotI+1µL of 6X loading dye
  • Well 5 : 5µL BBa_K592009 clone 2 digested by NotI+1µL of 6X loading dye
  • Well 6 : 5µL BBa_K592009 clone 1 digested by XhoI+1µL of 6X loading dye
  • Well 7 : 5µL BBa_K592009 clone 2 digested by XhoI+1µL of 6X loading dye
  • Well 8 : 5µL BBa_I732017 clone 1 digested by EcoRI/PstI+1µL of 6X loading dye
  • Well 9 : 5µL BBa_I732017 clone 2 digested by EcoRI/PstI+1µL of 6X loading dye
  • Well 10 : -
  • Well 11 : -
  • Well 12 : 6µL DNA Ladder
  • Gel : 1%

Expected sizes :

  • BBa_K592009 digested by NotI : 2046bp + 693bp
  • BBa_K592009 digested by XhoI : 1842bp + 892bp
  • BBa_I732017 : 3093bp

We obtain fragments at the right size. Our extraction was good.


Previous week Back to calendar Next day

Retrieved from "http://2013.igem.org/Team:Paris_Saclay/Notebook/July/8"