Team:Paris Bettencourt/Protocols

Ferment It Yourself

iGEM Paris-Bettencourt 2O15

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Protocols

Analytical digestion

Analytical digestion protocol

• Prepare the following mix:
  • 2µL 10X Digestion buffer
  • 0.5µL Eco31I
  • 0.5µL BbsI
  • 2µL of DNA (200ng)
  • 15µL water
• incube 1h at 37°C

Annealing

Annealing Protocol

• Phosphorylation of the oligos
  • 5.6μL DNAse/RNAse free water
  • 6.0μL oligo 1 (10µM)
  • 6.0μL oligo 2 (10µM)
  • 2.0μL 10X T4 DNA ligase buffer
  • 0.4μL T4 PolyNucleotide Kinase Total: 20μL
  
  
• incube 30min at 37°C
• add 1μL of 1M NaCl
• incube 5min at 95°C
• let the mix cool down
• use 2μL of the mix as a 10X solution

Electroporation

Electrocompetent Cells Preparation

Digestion

Electrocompetent Cells Preparation for Lactobacillus plantarum

Heat Shock transformation

Titration protocols for vitamin A

Yeast lysis with NaOH

Heat shock transformation for yeast

Ligation

Titration protocols for vitamin B2 with HPLC

Idli recipe

Miniprep protocol using a QIAGEN kit

PCR purification

PCR

Gel purification

Yeast DNA extraction using DNeasy® Blood & Tissue Kit [QIAGEN]

Titration of phytic acid using Megazyme© Kit

Gel Electrophoresis with SYBR safe