Purification

....

Restriction digest

For a restriction digest of 500ng DNA, you need

• Restriction enzymes
• Corresponding NEB buffer
• 100X BSA
• XμL DNA (500ng)
• 42.5-XμL dH₂O
• 0.6mL tube

Protocol:

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

...

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

...