Team:Paris Saclay/Notebook/August/13

From 2013.igem.org

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{|
{|
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
| style="border:1px solid black;padding:5px;background-color:#DEDEDE;" |
-
We can't see any band for Bba_K1155000 digestion. The digestion failed because we have used wrong enzymes. We will do it again with the good ones.
+
We can't see any band for Bba_K1155000 digestion. The digestion failed. We will do it again.
|}
|}
 +
=====2 - Digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI and by SpeI/EcoRI=====
 +
Anaïs, Nadia
 +
* Bba_K1155000 :
 +
** Buffer : 5µL
 +
** H2O : 39µL
 +
** DNA : 5µL
 +
** SpeI : 1µL
 +
*Bba_K1155000 :
 +
** Buffer : 5µL
 +
** H2O : 38µL
 +
** DNA : 5µL
 +
** SpeI : 1µL
 +
**EcoRI : 1µL
 +
*Bba_K1155004, Bba_K1155005, bba_K1155006 :
 +
** Buffer : 2µL
 +
** H2O : 7µL
 +
** DNA : 10µL
 +
** SpeI : 1µL
 +
 +
*Bba_K1155004, Bba_K1155005, bba_K1155006 :
 +
** Buffer : 2µL
 +
** H2O : 6µL
 +
** DNA : 10µL
 +
** SpeI : 1µL
 +
** EcoRI : 1µL
 +
 +
===='''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 :
 +
 +
[[File:PsPcr808.jpg|400px]]
 +
 +
=====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.
 +
 +
====3 - PCR product (made the 08/01/2013) purification====
 +
Damir
 +
 +
available quantity:
 +
* FNR Part1 : 10 µl
 +
* FNR Part2 : 19 µl
 +
* RBS FNR Part1 :16.1µl
 +
* RBS BphR2 Part1 : 28µl
 +
* BphR2 Part1 : 16.4 µl
 +
* BphR2 Part2 : 18.9 µl
 +
 +
Protocol : [[Team:Paris_Saclay/Protocols/kit_purification|kit purification]]
 +
 +
<span style="color:#FF5500;">Manipulation error :</span> The elution step was made using the recuperation tube from the filtering step, instead of a new, clean eppendorf tube.
   
   

Revision as of 09:54, 21 August 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 13

Lab work

A - Aerobic/Anaerobic regulation system

Objective : obtaining ...

1 - Electrophoresis of digestion of Bba_K1155000 by SpeI and PstI

Nadia

[[]]
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 5µL Bba_K1155000 digested by SpeI and PstI+1µl of 6X loading dye
  • Gel : 0.8%

Expected sizes :

  • Pfnr : ...
  • PSB1C3 : ...

We can't see any band for Bba_K1155000 digestion. The digestion failed. We will do it again.

2 - Digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI and by SpeI/EcoRI

Anaïs, Nadia

  • Bba_K1155000 :
    • Buffer : 5µL
    • H2O : 39µL
    • DNA : 5µL
    • SpeI : 1µL
  • Bba_K1155000 :
    • Buffer : 5µL
    • H2O : 38µL
    • DNA : 5µL
    • SpeI : 1µL
    • EcoRI : 1µL
  • Bba_K1155004, Bba_K1155005, bba_K1155006 :
    • Buffer : 2µL
    • H2O : 7µL
    • DNA : 10µL
    • SpeI : 1µL
  • Bba_K1155004, Bba_K1155005, bba_K1155006 :
    • Buffer : 2µL
    • H2O : 6µL
    • DNA : 10µL
    • SpeI : 1µL
    • EcoRI : 1µL

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 :

PsPcr808.jpg

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.

3 - PCR product (made the 08/01/2013) purification

Damir

available quantity:

  • FNR Part1 : 10 µl
  • FNR Part2 : 19 µl
  • RBS FNR Part1 :16.1µl
  • RBS BphR2 Part1 : 28µl
  • BphR2 Part1 : 16.4 µl
  • BphR2 Part2 : 18.9 µl

Protocol : kit purification

Manipulation error : The elution step was made using the recuperation tube from the filtering step, instead of a new, clean eppendorf tube.




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