Team:Czech Republic/Protocols

Protocols

Purification

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Restriction

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Band-stab PCR

Excellent technique when you want to amplify specific band from a gel. http://bitesizebio.com/13512/pcr-rescue/ More info..

  • Prepare a complete 50μL PCR reaction without DNA template
  • Take a sterile pipette tip and stab the desired band of interest 2-3 times
  • Swirl the tip in the tube with prepared PCR reaction
  • Run the PCR as usual

GA

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Soft lithography - PDMS molding

  • Mix 40g of PDMS with 4g of curing agent
  • Centrifuge the mixture at 3250 RPM for 3 minutes to remove the bubbles introduced during the mixing
  • Clean silicon master with air gun
  • Wrap an aluminium foil around the edges of the silicon master to create a container
  • Pour the PDMS mixture over the silicon master
  • Cure the PDMS in an oven at 80°C for 2 hours
  • Leave the PDMS to cool down
  • Detach the PDMS carefully from the silicon master

Soft lithography - Bonding PDMS-Glass

Preparation of the substrates

Glass slide
  • Rinse the glass slide with acetone, isopropanol, and deionized water
  • Dry the glass slide with air gun
  • Dehydrate the glass slide on a hot plate at 120°C for 30 minutes
PDMS
  • Slice the PDMS to individual PDMS replicas
  • Drill holes for microfluidic inlets and outlets
  • Clean the PDMS using a scotch tape

Air plasma treatment

  • Place the glass slide and the PDMS replicas into a plasma cleaner, contact surfaces facing upwards
  • Exhaust the atmosphere with a vacuum pump and wait until the pressure drops to 500 mTorr
  • Activate the plasma for 2.5 minutes at Hi power
  • Stop the plasma and open the valve to stabilize the pressure, continue immediately with the bonding phase

Permanent irreversible bonding

  • Place the PDMS replica on the glass slide
  • Place the bonded device in an oven at 80°C for 60 minutes.
  • Seal the inlets and outlets with a scotch tape until use

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