Team:Paris Bettencourt/Protocols

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Electroporation
Analytical digestion
Electrocompetent Cells Preparation
Heat Shock transformation
Ligation
Digestion
Miniprep protocol using a QIAGEN kit
Annealing
PCR purification

Electroporation Protocol Thaw electrocompetent cells on ice Add 2µL of ligation product or 0.5µL of native plasmid to the cells Transfer the cells in an 0.2mm electroporation cuvette put the cuvette in the electroporation device and pulse the cells at 2.5kV, 200 Ohms and 25µF add 200µL of LB right after pulsing recover 2 hours at 37°C plate 200µL and 50µL of the cells on LB + erythromycin (200µg/mL), incubate overnight at 37°C

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

Electrocompetent Cells Preparation Protocol Inoculate two 250mL LB flasks with 100µL of an overnight culture of DH5α incubate until the the DO600 reach 0.5 to 0.7 place the cultures on ice for 15 minutes pour the culture in cold sterile 50mL falcon tubes centrifuge them for 10 minutes at 6000rpm throw the supernatant resuspend the cells in 50mL cold distilled water centrifuge them for 10 minutes at 6000rpm throw the supernatant resuspend the cells in 25mL cold distilled water centrifuge them for 10 minutes at 6000rpm throw the supernatant resuspend the cells in 12.5mL cold 10% glycerol centrifuge them for 10 minutes at 6000rpm throw the supernatant resuspend the cells in 5mL cold 10% glycerol make aliquots of the desire volume in microcentrifuge tubes and freeze them at -80°C

Heat Shock transformation protocol Thaw frozen chemically competent cells (20µL aliquots) on ice for 10min. add 2µL of ligation product (or 0.5µL of miniprep) and incubate the cells 30sec at 42°C. put the cells back on ice for 2min. add 200µL of LB to the cells and incubate 2 hours at 37°C. plate the cells on LB + erythromycin (150µg/mL) or LB + erythromycin (10µg/mL), incubation at 37°C overnight.

Ligation Protocol Mix the following vector 100ng insert 300ng T4 DNA ligase buffer 10X T4 DNA ligase up to 20µL of water incubate 30 to 40 minutes at room temperature

Digestion Protocol Prepare the following mix: 4μL of Enzyme 1 4μL of Enzyme 2 4μL of FastAP 12μL of Fast Digest buffer 10X 1 to 3 μg of DNA up to 120μL of water mix by pipetting up and down incube 10min at 37°C

Miniprep protocol using a QIAGEN kit centrifuge an overnight culture of cells 10min at 4krpm throw the filtrate resuspend the pellet in 250μL of Cell Resuspension Solution then mix it transfer it in a 1.5mL microcentrifuge tube add 250mL of Cell lysis solution and mix by inverting several times incube until the liquid is clear, maximum 5min add 10μL of Alkalyne protease solution, mix by inverting several times and incube 3 to 4 min add 350μL of Neutralisation Solution and mix by inverting several times centrifuge 10min at 14krpm add the supernatant in a column centrifuge 1min, 14krpm, then throw the filtrat add 750μL Washing Solution centrifuge 1min, 14krpm, then throw the filtrat add 250μL Washing Solution centrifuge 2min, 14krpm, then throw the filtrat centrifuge 1min, 14krpm transfer the column in a sterile microcentrifuge 1.5ml tube add 50μL of DNAse/RNAse free water right on the membrane of the filter, wait 1min centrifuge 1min, 14krpm throw the column, plasmid is saved in water

Annealing Protocol Phosphorylation of the oligos 5.6μL DNAse/RNAse free water 6.0μL o15.011 (10µM) 6.0μL o15.012 (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

PCR purification protocol Add 5 volumes of resuspension buffer to 1 volume of PCR product in an 1.5mL microcentrifuge tube, mix by pipetting up and down Transfer in a centrifugation column Centrifuge 1 min at 14000 rpm Throw the filtration product Add 700μL of washing solution Centrifuge 1 min at 14000 rpm Throw the filtration product Add 500μL of washing solution Centrifuge 1 min at 14000 rpm Throw the filtration product Centrifuge 1 min at 14000 rpm Throw the filtration product Put the column in a sterile 1.5mL microcentrifuge tube Add 45μL of DNAse/RNAse free water on the membrane Wait 2 minutes Centrifuge 2min at 10000rpm Discard the column, DNA is saved in water