Team:UCLouvain/Experiments

 Experiments


Miniprep

Miniprep were done with Sigma GenElute Plasmid Miniprep Kit See protocol at https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/pln70bul.pdf

Enzyme digestion protocols

Digestion were done with NEB (NewEngland BioLabs) enzymes, according to the official protocol See https://nebcloner.neb.com/#!/redigest
Classical enzyme digestion for 50 µl reaction:
  • DNA 1 µg
  • 10X Buffer 5 µl (1X)
  • Enzyme 1 1.0 µl
  • Enzyme 1 1.0 µl
  • Nuclease-free Water Up to 50 µl
  • Incubate at 37°C for 5-15 minutes

PCR protocol

PCR were done with the Q5 DNA Polymerase (from NEB) according to the official protocol.
image

Ligation protocol

Ligation were done with T4 Ligase (Roche)
image

Phenol/chloroform purification protocol

  • add in an Eppendorf tube 20 µl DNA + 380 µl H2O (volume = 400 µl)
  • add 1:1 volume of phenol/chloroform, vortex, centrifugation 13500 rpm during 2 minutes
  • transfer supernatant in a new tube
  • add one volume of chloroform, vortex, centrifugation 13500 rpm during 2 minutes
  • add 0,2 volume of 10M ammonium acetate + 2,5 volume of cold ethanol (100%), invert tube, and put at -20°C during 30 minutes
  • Centrifugation at 13500 rpm during 5 minutes, discard the supernatant
  • Wash 2 times with 1 ml cold ethanol (70%) and centrifuge 5 minutes at 4°C
  • Discard the supernatant
  • Quick-spin, remove ethanol
  • Keep open the tube during 20 minutes to evaporate the ethanol
  • add 20 µl H2O MilliQ, water bath at 37°C for 15 to 30 minutes
  • Quick-spin, resuspend DNA and verification of the concentration (Nanodrop)

Transformation with electro-competant cells

  • Prepare in a 15ml falcon, 250 ul of SOC medium
  • Defrost 50 µl aliquot of electrocompetant cells
  • add about 1 µl (less then 1 ng) of plasmidic DNA into the 50 µl of the competent cells
  • Place it into an electroporation cuvette
  • Programm the Bio-Rad to pulse 2500 V
  • electroporate
  • add the 250 µl of SOC and put back in the falcon
  • place the falcon at 37°C for one hour
  • put 20 µl and 100 µl of the electroporated cells on a LB plate with antibiotics
  • incubate overnight

Electrophoresis gel protocols

Agarose gel used of 0,8%, in BioRad syste

Glycerol stock

Glycerol used at 25%

Conjugation “in plate” protocol

Day 1

  • Pick up a single colony from donnor, control and recipient strain, grown overnight on LB plates and inoculate in 5 ml of LB with respective antibiotics.
  • Incubate overnight at 37°C.

Day 2

  • Centrifuge the preculture 5 minutes at 4000 rpm
  • Remove the supernatant ( optional : wash 2 times with 1 ml of PBS)
  • Resuspend the pellets in 3 ml of LB (donnor in excess increase the conjugation efficiency)
  • Incubate 1 hour at 37°C to regenerate the pili
  • On LB agar plate, put 5 µl of donnor, control, recipient strain, and put 5µl of recipient + 5 µl of donor strain on top of the receiving strain ( see picture)
    image
  • Incubate at 37°C between 2 and 16 hours
  • Scrap the cells and resuspend in 200 µl of LB
  • Plate each condition in the adequate LB plate and incubate overnight at 37°C
  • Conjugation efficiency = recipient cells that received the plasmid / recipient cells

Proteinase K protocol

  • Add 0,2 volume of ProteinaseK (300 ug/ml) (final 50ul) to DNA
  • Incubate 1 hour at 37°C
  • Incubate in water-bath at 75°C for 25 minutes for Proteinase K inactivation

Resuspension of insert synthetic

  • centrifuged the synthetic DNA during 5 seconds at 300 g
  • add 25µl of water miliQ
  • incubated during 20 minutes at 50°C
  • quick spin and resuspend the DNA

DNA purification with Amicon® Ultra-0.5 Centrifugal Filter Devices

  • Insert the Amicon® Ultra-0.5 device into one of the provided microcentrifuge tubes.
  • Add up to 500 μL of sample to the Amicon® Ultra filter device and cap it.
  • Place capped filter device into the centrifuge rotor, aligning the cap strap toward the center of therotor; counterbalance with a similar device.
  • Spin the device at 14,000 × g for approximately 10–30 minutes depending on the NMWL of the device used. Refer to Figure 1 and Table 2 for typical spin times.
  • Spin the device at 14,000 × g for approximately 10–30 minutes depending on the NMWL of the device used. Refer to Figure 1 and Table 2 for typical spin times.
  • To recover the concentrated solute, place the Amicon® Ultra filter device upside down in a clean microcentrifuge tube. Place in centrifuge, aligning open cap towards the center of the rotor; counterbalance with a similar device. Spin for 2 minutes at 1,000 × g to transfer the concentrated sample from the device to the tube. The ultrafiltrate can be stored in the centrifuge tube.