Team:Paris Saclay/Notebook/August/8

From 2013.igem.org

(Difference between revisions)
Line 105: Line 105:
|}
|}
 +
===='''Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006'''====
 +
===='''1 - Tranduction of Km in MG1655Z1 ====
 +
 +
Anaïs, Nadia
 +
 +
{|
 +
| style="border:1px solid black;padding:5px;background-color:#DE;" |
 +
We didn't obtain colonies from the transduction of 08/07/13. We will do it again.
 +
|}
 +
 +
Protocol : [[Team:Paris_Saclay/Protocols/transduction|Transduction]]
 +
 +
Our Mutant bacteria was called BW : Δfnr::Km.
 +
Our wild type bacteria was called MG1655Z1.
 +
 +
We did the first step of the protocol : bacteriophage stock which packed Km gene. 
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===
==='''A - Aerobic/Anaerobic regulation system / B - PCB sensor system'''===

Revision as of 14:35, 28 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Contents

Notebook : August 8

Lab work

A - Aerobic/Anaerobic regulation system

Obtaining the PSB3K3 backbone plasmid

1 - Electrophoresis of BBa_J004450 digested by EcoRI/Pst1 to check if the digestion

Damir, Nadia

IMAGE
  • 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 obtain fragments at the right size. We will purify the highest band which contains the PSB3K3 plasmid.

2 - Electrophoresis of Bba_J004450 digested by EcoRI/PstI

Anaïs

PsNBa8 eelution.jpg
  • Well 1 : 6µL DNA Ladder
  • Well 2 : 45µL of BBa_J004450 digested by EcoRI/Pst1
  • Gel : 0.8%

We obtain fragments at the right size. We will purify the highest band which contains the PSB3K3 plasmid.

3- Electroelution of PSB3K3 digested by EcoRI/PstI

Nadia

Protocol : Electroelution

We let the plasmid precipitate during the night.

Objective : obtaining Bba_K1155007

1 - Colony PCR of Bba_K115007 in DH5α

Anaïs

Tranformation of 08/07/13 works. we will make a PCR Colony.

Colonie repiquée dans 10µL d'eau pour chaque tube ???????

Used quantities :

  • DNA : 2µL
  • Mix  : (it was divided in 25 tubes for each promotor with 23µL of mix in each on)
    • Oligo ... : 3.5µL
    • Oligo ... : 3.5µL
    • Buffer Dream Taq : 70µL
    • dNTP : 28µL
    • Dream Taq : 5µL
    • H2O : 590µL

PCR Program :

PsPcr808.jpg

2 - Electrophoresis to check the colony PCR products : Bba_K1155007

Anaïs, Damir

350px
  • Well 1 : 6µL DNA Ladder
  • Well 2 to 24 : 10µL of Bba_K1155007+2µL of 6X loading dye
  • Well 25 : 6µL DNA Ladder
  • Well 26 : 6µL DNA Ladder
  • Well 27 : 10µL of Bba_K1155007+2µL of 6X loading dye
  • Gel : 0.8%

Expected size :

  • Bba_K1155007 : 3583 bp

We obtain fragment at the right size for colonies 10, 14 and 15. We will extract BBa_K1155007.

Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006

1 - Tranduction of Km in MG1655Z1

Anaïs, Nadia

We didn't obtain colonies from the transduction of 08/07/13. We will do it again.

Protocol : Transduction

Our Mutant bacteria was called BW : Δfnr::Km. Our wild type bacteria was called MG1655Z1.

We did the first step of the protocol : bacteriophage stock which packed Km gene.

A - Aerobic/Anaerobic regulation system / B - PCB sensor system

Objective : obtaining FNR and BphR2 proteins

1 - Extraction of plasmid of BphR2, FNR, RBS-FNR

Damir

Transformation of 08/02/13 works. We will extract plasmids.

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

We lost our plasmids. We will do the Gibson assembly again.

2 - Gel purification of RBS-BphR2 Part I

Nadia, XiaoJing

Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]

We lost fragment. We will do the PCR again.


Previous day Back to calendar Next day

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