Team:Czech Republic/Protocols

Protocols

Purification

  • Nucleospin Gel and PCR clean-up
  • 1.5ml eppendorf tube
  1. Mix 1 volume of sample with 2 volumes of NTI buffer (for example 100ul of sample with 200ul of NTI buffer) or for each 100mg of agarose gel add 200ul Buffer NTI and incubate the gel-sample for 5 – 10min at 50C and vortex until the gel slice is completely dissolved.
  2. Transfer at max 700ul of sample into the spin column and centrifuge 30s at 11,000g (for sample from gel is maybe necessary to repeat this and next step).
  3. Discard the flow-through.
  4. Add 700uL of NT3 buffer, centrifuge 1min at 11,000g.
  5. Discard the flow-through.
  6. Centrifuge empty column 3min at 11,000g.
  7. Discard the flow-through and place the spin column into eppendorf tube.
  8. Put the tube in shaker pre-heated on 70 C and incubate for 3min at 70 C.
  9. Instil 15uL of Elution buffer into the center of spin column filter.
  10. Incubate for 3min at room temperature or 70 C if the DNA fragments is longer than 1000 bp.
  11. Centrifuge 1min at 11,000g.
  12. Repeat again steps 9-11 .

Transformation (bacteria)

Transformation (yeast)

Gel electrophoresis

TBE stock 10X (1000ml)

  • 108g Trisbase
  • 55g Boric acid
  • 9.3g EDTA (Ethylenediaminetetraacetic acid)
  • dH20
  1. Add the ingredients into 900ml of dH20, mix it properly and fill to 1000ml.
  2. Store at room temperature.

NaOH stock 0,1M (200mL)

  • 0.8g NaOH
  • Boric acid
  • dH20
  1. Add the ingredients into 200ml of dH20.
  2. Set pH on 8,5 by Boric acid.
  3. Store at room temperature.

Electrophoresis gel (100ml) 1%

  • Agarose
  • 1X TBE or 0,1M NaOH
  • Ethidium bromide

Protocol:

  1. Add 0,7g agarose to 100ml 1X TBE or 10mM NaOH.
  2. Heat in microwave for 1 min at high power.
  3. Mix it and put it again into microwave for 1 min at high power.
  4. Make sure you have nitril gloves.
  5. Let it chill down for 6min and add 4 uL of ethidium bromide.
  6. Pour the balanced plate, don't forget to put the rake on.
  7. After the gel congeal, cover it with 1X TBE or 10mM NaOH.

For making small electrophoresis use half the amounts.

Miniprep

  • Miniprep NucleoSpin Plasmid
  • 2x 1.5ml eppendorf tube

Protocol:

    • Overnight culture: Centrifuge 5ml at 11,000g for 30s, remove the liquid, add 250 uL A1 buffer, vortex properly and transfer it to the eppendorf tube.
    • Petri dish: Add 250 L A1 buffer to the eppendorf tube, scrape off and add a culture from one partition, vortex properly.
  2. Add 250 uL A2 buffer (blue), gently invert 8 times, incubate for 5min at room temperature.
  3. Add 300 uL A3 buffer, gently invert to the change of colour (from blue to white).
  4. Centrifuge 7min at 11,000g.
  5. Transfer max 750 uL of the supernatant into 2ml spin column.
  6. Centrifuge 1min at 11,000g.
  7. Discard the flow-through and add 600 uL A4 buffer, centrifuge 1min at 11,000g.
  8. Discard flow-through and centrifuge 3min at 11,000g.
  9. Put the spin column into new eppendorf tube.
  10. Instil 25 uL DNA free water into the center of spin column filter, incubate for 3min at room temperature.
  11. Centrifuge for 1min at 11,000g.
  12. Repeat again steps 10-12.

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

DNA Ligation

Insert mass in ng = \(3\frac{Insert\,length\,in\,bp}{Vector\,length\,in\,bp}Vector\,mass\,in\,ng\)

  • 10X T4 ligase buffer
  • 0.5uL T4 ligase
  • Purified insert and vector (~50ng vector) DNA
  • (8.5 - vector and insert)uL ultrapure dH20
  • 0.6mL tube

Protocol:

  1. Add appropriate amount of ultrapure water to sterile 0.6mL tube.
  2. Vortex 10X T4 ligase buffer and add 1uL to the tube.
  3. Add appropriate amount of vortexed insert and vector DNA to the tube.
  4. Vortex T4 ligase and add 0.5uL to the tube.
  5. Place the tube in thermal cycler and run ligation protocol (60min incubation at 16°C, 10min at 65°C to denaturate the ligase)
  6. Store at -20°C

PCR Methods

Preparation of Primers

  1. Centrifuge a primer in the original tube.
  2. For 1 nmol of primer add 10 ul of DNA free H2O into original tube and vortex properly.
  3. Into new eppendorf tube add 24 ul of DNA free H2O, 3 ul of forward primer and 3 ul of reverse primer – work concentration

One Taq PCR protocol (25 ul reaction)

  1. Into PCR tube add 17,875ul of dH2O
  2. Add 5ul 5X One Taq Standard reaction buffer, 0,5ul dNTPs, 0,5ul of mix primers – work concentration, 1ul template DNA – work concentration 1 ng/ul and 0,125ul One Taq DNA polymerase.

Q5 PCR protocol

  1. Into PCR tube add 17 ul DNA free H2O.
  2. Add 5 ul 5X Q5 reaction buffer, 0,5 ul dNTPs, 1,25 ul of mix primers – work concentration, 1 ul template DNA – work concentration 1 ng/ul and 0,25 ul Q5 High-Fidelity DNA polymerase.

Adjustement of thermo cycler

  • Denaturation – 10s, 98°C
  • Annaeling - 30s, temperature depends on the primers
  • Extension – time depends on the length of PCR construct - 30s for 1000bp, 68° for Taq / 72° for Q5

Band-stab PCR

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

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

Gibson

Genome extraction

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