Paris/July 20

From 2007.igem.org

< Paris(Difference between revisions)
 
(12 intermediate revisions not shown)
Line 1: Line 1:
 +
[[Paris/July 19|yesterday]] -- [[Paris/July 21|tomorrow]] <br>
==PCRs==
==PCRs==
-
 
-
 
-
'''Je n’ai pas pu lancer la PCR car :
 
-
 
-
Les oligos O10 et O11 ne sont pas dilués à 10μM (ni les autres oligos après O9 d’ailleurs ??!!). Les fiches des oligos ne sont pas dans le classeur, je ne peux donc pas savoir à quelle concentration est le stock !'''
 
-
NB : Tous les oligos en stock sont à 100µM. Et ils sont ensuite dilués à 10µM dans l'autre boïte
 
-
 
{{Paris_PCR_0| Title = Lox71-KmR-Lox66
{{Paris_PCR_0| Title = Lox71-KmR-Lox66
-
|Annealing= 55°(3x)-63°(35x)C
+
|Annealing= 55
-
|Elongation= 2m00'
+
|Elongation= 3m00'
-
|Cycles= 38
+
|Cycles= 35
|Buffer=  5x 10µL
|Buffer=  5x 10µL
|MgCl2= 10µM 0µL
|MgCl2= 10µM 0µL
Line 19: Line 13:
|n_oligoR= 11  Lox66-KmR-R
|n_oligoR= 11  Lox66-KmR-R
|v_oligoR= 2.5µL
|v_oligoR= 2.5µL
-
|water= 30µL
+
|water= 34µl
|pol= Phusion 0.5µL
|pol= Phusion 0.5µL
-
|DNA= MP3_pSB1AK3-BBa_B0015 2µL dil.100
+
|DNA= MP3_pSB1AK3-BBa_B0015 0.5µl
-
|Size=
+
|Size= 1084
-
|Success=
+
|Success= ?
 +
|Image=
 +
|Band=
 +
|}}
 +
 
 +
{{Paris_PCR_0| Title = Lox71-FtsA-FtsZ
 +
|Annealing= 55
 +
|Elongation= 3m00'
 +
|Cycles= 35
 +
|Buffer=  5x 10µL
 +
|MgCl2= 10µM 0µL
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= 3 Lox71-FtsA-F
 +
|v_oligoF= 2.5µL
 +
|n_oligoR= 2 FtsZ-R
 +
|v_oligoR= 2.5µL
 +
|water= 34µl
 +
|pol= Phusion 0.5µL
 +
|DNA= toothpick in MG1655 glycerol
 +
|Size= 2588
 +
|Success= ? The PCR seemed to have worked but the purification failed
|Image=
|Image=
|Band=
|Band=
Line 30: Line 44:
==Glycerol stock==
==Glycerol stock==
-
* BBa_pJ23107 clone 3&4
+
* BBa_pJ23107 clone 3&4 (S14)
-
* BBa_I0500 clone 1&2
+
* BBa_I0500 clone 1&2 (S13)
==Minipreps on Biobricks==
==Minipreps on Biobricks==
Line 37: Line 51:
* BBa_pJ23107 clone 3&4
* BBa_pJ23107 clone 3&4
* BBa_I0500 clone 1&2
* BBa_I0500 clone 1&2
 +
 +
== E.Coli pKS::DGAT ==
 +
 +
We look under microscopy 36 hours after incubation of E.coli transformed by pKS::DGAT and the control E.coli transformed by part B0015  on different LB medium (See [[Paris/July_18#E.coli_pKS::DGAT|July 18]]).
 +
 +
* Observations:
 +
We can observe colonies (X10).
 +
[[Image: coli_dgat_07202007.jpg|center|900px]]
 +
 +
* Interpretation
 +
 +
We can observe lipid inclusions within colonies with DGAT. Further investigations need single cell visualisation.

Latest revision as of 17:49, 7 October 2007

yesterday -- tomorrow

Contents

PCRs

PCR : Lox71-KmR-Lox66
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 1084
55 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 10 Lox71-KmR-F 2.5µL  ?
3m00' Oligo R 10µM 11 Lox66-KmR-R 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
35 Polymerase Phusion 0.5µL Band (0=ladder)
DNA MP3_pSB1AK3-BBa_B0015 0.5µl
PCR : Lox71-FtsA-FtsZ
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 2588
55 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 3 Lox71-FtsA-F 2.5µL  ? The PCR seemed to have worked but the purification failed
3m00' Oligo R 10µM 2 FtsZ-R 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
35 Polymerase Phusion 0.5µL Band (0=ladder)
DNA toothpick in MG1655 glycerol

Glycerol stock

  • BBa_pJ23107 clone 3&4 (S14)
  • BBa_I0500 clone 1&2 (S13)

Minipreps on Biobricks

  • BBa_pJ23107 clone 3&4
  • BBa_I0500 clone 1&2

E.Coli pKS::DGAT

We look under microscopy 36 hours after incubation of E.coli transformed by pKS::DGAT and the control E.coli transformed by part B0015 on different LB medium (See July 18).

  • Observations:

We can observe colonies (X10).

Coli dgat 07202007.jpg
  • Interpretation

We can observe lipid inclusions within colonies with DGAT. Further investigations need single cell visualisation.

Retrieved from "http://2007.igem.org/wiki/index.php/Paris/July_20"