Difference between revisions of "Team:Czech Republic/Protocols"
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= Soft lithography - Bonding PDMS-Glass = | = Soft lithography - Bonding PDMS-Glass = | ||
| − | + | == Preparation of the substrates == | |
| − | + | === Glass slide === | |
*Rinse the glass slide with acetone, isopropanol, and deionized water | *Rinse the glass slide with acetone, isopropanol, and deionized water | ||
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*Dehydrate the glass slide on a hot plate at 120°C for 30 minutes | *Dehydrate the glass slide on a hot plate at 120°C for 30 minutes | ||
| − | + | === PDMS === | |
*Slice the PDMS to individual PDMS replicas | *Slice the PDMS to individual PDMS replicas | ||
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*Clean the PDMS using a scotch tape | *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 | *Place the glass slide and the PDMS replicas into a plasma cleaner, contact surfaces facing upwards | ||
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*Stop the plasma and open the valve to stabilize the pressure, continue immediately with the bonding phase | *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 PDMS replica on the glass slide | ||
Revision as of 16:06, 16 September 2015
Protocols
Contents
Purification
....
Restriction digest
For a restriction digest of 500ng DNA, you need
- Restriction enzymes
- Corresponding NEB buffer
- 100X BSA
- XμL DNA (500ng)
- (42.5-X)μL dH2O
- 0.6mL tube
Protocol:
- Add appropriate amount od dH2O to the tube
- Vortex NEB buffer and add 5μL
- Vortex BSA and add 0.5μL (no need to add BSA when using NEB CutSmart buffer)
- Vortex DNA and add appropriate amount to the tube
- Vortex each enzyme and add 1μL to the tube
- Incubate at 37°C for 30min, then heat-inactivate at 80°C for 20min
- Store at 4°C
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
...