Team:Paris Saclay/Notebook/August/9

From 2013.igem.org

(Difference between revisions)
(Created page with "{{Team:Paris_Saclay/incl_debut_generique}} ='''Notebook : August 9'''= =='''Lab work'''== ==='''A - Aerobic/Anaerobic regulation system'''=== ===='''Obtaining Δ ''fnr E. col...")
Line 9: Line 9:
===='''Obtaining Δ ''fnr E. coli'' strain by transduction to test our biobricks'''====
===='''Obtaining Δ ''fnr E. coli'' strain by transduction to test our biobricks'''====
-
=====1 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion was right=====
+
Abdou, Anais, Damir, Nadia, XiaoJing
-
Damir, Nadia
+
-
{|
+
We do the exprience again because the lysis made on wensday didn't happen. It's probably because the phage strain  was too old ( 2001) 
-
| style="width:350px;border:1px solid black;" | IMAGE
+
-
| style="width:350px;border:1px solid black;vertical-align:top;" |
+
-
*Well 1 : 6µL DNA Ladder
+
-
*Well 2 : 5µL of BBa_J004450 digested by EcoRI/Pst1
+
-
*Gel : 0.8%
+
-
|}
+
-
 
+
-
Expected sizes :
+
-
 
+
-
*PSB3K3 : 2750kb
+
-
*GFP : 1069kb
+
-
 
+
-
We obtained fragments of the right size. Now we can purify the PSB3K3 plasmid.
+
-
 
+
-
=====2 - Gel purification of the PSB3K3 plasmid from BBa_J004450 digested by EcoRI/Pst1=====
+
-
 
+
-
''2.1 - Migration of the remaining 45µL of BBa_J004450 digested by EcoRI/Pst1''
+
-
Anaïs
+
Protocol : [[Team:Paris_Saclay/Protocols/Colony_PCR|Colony PCR]]
{|
{|
-
| style="width:350px;border:1px solid black;" | [[File:PsNBa8_eelution.jpg|350px]]
+
| style="width:350px;border:1px solid black;" | [[File:Ps908transduction.jpg|350px]]
| style="width:350px;border:1px solid black;vertical-align:top;" |
| style="width:350px;border:1px solid black;vertical-align:top;" |
*Well 1 : 6µL DNA Ladder
*Well 1 : 6µL DNA Ladder

Revision as of 11:31, 11 August 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 9

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining Δ fnr E. coli strain by transduction to test our biobricks

Abdou, Anais, Damir, Nadia, XiaoJing

We do the exprience again because the lysis made on wensday didn't happen. It's probably because the phage strain was too old ( 2001)

Protocol : Colony PCR

Ps908transduction.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 45µL of BBa_J004450 digested by EcoRI/Pst1
  • Gel : 0.8% 
Now we can do a gel purification of the highest band which contains the PSB3K3 plasmid.

2.2 - Electroelution of the highest band to extract the PSB3K3 plasmid from the gel

Nadia

Protocol : Electroelution 

We let the plasmid precipitate during the night.

Obtaining RBS_LacZ+Term_PSB1C3

1 - Colony PCR on e.coli with RBS_LacZ+Term_PSB1C3 for 25 colonies

Anaïs

  • Colony counting :
    • Low concentration petri dish : 47 colonies
    • High concentration petri dish : 145 colonies
  • Picking of 25 colonies
  • Preparation of 700µL of Master mix
    • H2O : 590µL
    • dNTP : 28µL
    • VF2 primer : 3.5µL
    • VR primer : 3.5µL
    • DreamTaq buffer 10x : 70µL
    • DreamTaq enzyme : 5µL

Protocol : Colony PCR 

PCR Program :
 [IMAGE]
2 - Gel electrophoresis of the colony PCR products

Anaïs, Damir

PsNBa8 colonies.jpg
  • 6µL DNA Ladder
  • 10µL sample per well
  • Gel : 0.8%

Expected size : 3583bp 

Colonies 10, 14, 15 exhibit plasmids with the right length.

Retrieved from "http://2013.igem.org/Team:Paris_Saclay/Notebook/August/9"