Difference between revisions of "Team:Czech Republic/Protocols"

(→‎Restriction)
(→‎Restriction digest)
Line 12: Line 12:
 
* 100X BSA
 
* 100X BSA
 
* XμL DNA (500ng)
 
* XμL DNA (500ng)
−
* 42.5-XμL dH<sub>2</sub>O
+
* (42.5-X)μL dH<sub>2</sub>O
 
* 0.6mL tube
 
* 0.6mL tube
  
 
Protocol:
 
Protocol:
  
−
#
+
# Add appropriate amount od dH<sub>2</sub>O 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 =
 
= Band-stab PCR =

Revision as of 13:45, 16 September 2015

Protocols

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:

  1. Add appropriate amount od dH2O to the tube
  2. Vortex NEB buffer and add 5μL
  3. Vortex BSA and add 0.5μL (no need to add BSA when using NEB CutSmart buffer)
  4. Vortex DNA and add appropriate amount to the tube
  5. Vortex each enzyme and add 1μL to the tube
  6. Incubate at 37°C for 30min, then heat-inactivate at 80°C for 20min
  7. 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

...