Team:Paris Saclay/Notebook/August/8

From 2013.igem.org

Revision as of 14:51, 11 August 2013 by Anais.D (Talk | contribs)

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 was right

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 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

PsNBa8 eelution.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 :

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.