Team:Paris Saclay/Notebook/August/9

From 2013.igem.org

(Difference between revisions)
Line 18: Line 18:
| style="width:350px;border:1px solid black;" | [[File:Ps908transduction.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;" |
-
'''picture: lysed cells comparison.'''
+
'''Picture: lysed cells comparison.'''
-
* 0µl phage(control): the petri dish is cloudy, bacteria are not lysed  
+
* 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.
* 50µl phage: the petri dish is clear, bacteria are lysed by phages.  
* 50µl phage: the petri dish is clear, bacteria are lysed by phages.  
*We used a wild type strain to keep a stock and a Δ ''fnr E. coli'' strain to obtain phages that might have encapsidated a Δfnr::Km fragment.  
*We used a wild type strain to keep a stock and a Δ ''fnr E. coli'' strain to obtain phages that might have encapsidated a Δfnr::Km fragment.  
|}
|}
-
  Now we can do a gel purification of the highest band which contains the PSB3K3 plasmid.
+
10µl,50µl and 100µl petri dishes are clear so phages are multiplied.
 +
   
 +
We let the antibiotic over night to select the right strain.  
-
''2.2 - Electroelution of the highest band to extract the PSB3K3 plasmid from the gel''
 
-
 
-
Nadia
 
-
 
-
Protocol : [[Team:Paris_Saclay/Protocols/Electroelution|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
 
-
**H<sub>2</sub>O : 590µL
 
-
**dNTP : 28µL
 
-
**VF2 primer : 3.5µL
 
-
**VR primer : 3.5µL
 
-
**DreamTaq buffer 10x : 70µL
 
-
**DreamTaq enzyme : 5µL
 
-
 
-
Protocol : [[Team:Paris_Saclay/Protocols/Colony_PCR|Colony PCR]]
 
-
 
-
PCR Program :
 
-
  [IMAGE]
 
-
 
-
=====2 - Gel electrophoresis of the colony PCR products=====
 
-
Anaïs, Damir
 
-
 
-
{|
 
-
| style="width:350px;border:1px solid black;" | [[File:PsNBa8_colonies.jpg|350px]]
 
-
| style="width:350px;border:1px solid black;vertical-align:top;" |
 
-
*6µL DNA Ladder
 
-
*10µL sample per well
 
-
*Gel : 0.8%
 
-
|}
 
-
 
-
Expected size : 3583bp
 
-
 
-
Colonies 10, 14, 15 exhibit plasmids with the right length.
 
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Revision as of 12:26, 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 : transduction

Ps908transduction.jpg

Picture: lysed cells comparison.

  • 0µl phage(control): the petri dish is cloudy, bacteria are not lysed.
  • 50µl phage: the petri dish is clear, bacteria are lysed by phages.
  • We used a wild type strain to keep a stock and a Δ fnr E. coli strain to obtain phages that might have encapsidated a Δfnr::Km fragment.

10µl,50µl and 100µl petri dishes are clear so phages are multiplied.

We let the antibiotic over night to select the right strain.


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