Team:EPF Lausanne/Test
Protocols
Agarose Gel
Materials
• 1X TAE
• Agarose
• Gel Red
• DNA samples
• 6X loading dye
• Nuclease free water
Procedure
Prepare 1.2% agarose gel for small fragments and 3% agarose gel for large fragments
• Mix 50 mL 1X TAE and 0.6 g (1.2%) or 1.5 g (3%) agarose
• Melt in microwave until agarose has melted (about 50 seconds)
• Add 1.3 μL (1.2%) or 1.5 μL (3%) Gel Red
• Pour solution into agarose gel mold with comb
• Let set for 20 minutes or until solid
• Place gel in 1X TAE and remove comb
• Load samples of 200 ng (or 2 μL) DNA mixed with 2 μL 6X loading dye and nuclease free water up to 12 μL
• Run gel at 100-120 Volts for 40-50 minutes (1.2%) or 80 Volts for 2 hours (3%)
• Take a picture of the gel at the UV detector
Amino acid solution
Materials
• Histidine-Hcl
• Uracil
• Leucine
• Tryptophan
Procedure
| Stock concentration | Final concentration | Total quantity for 50 mL |
|---|---|---|
| 100 mM Histidine-Hcl (209 g/mol) | 20.9 g/L | 0.418 g |
| 20 mM Uracil (112 g/mol) | 2.24 g/L | 0.0448 g |
| 100 mM Leucine (131 g/mol) | 13.1 g/L | 0.262 g |
| 40 mM Tryptophan (204 g/mol) | 8.16 g/L | 0.1632 g |
• Filter and sterilize solutions
• Add 8 mL per liter of selective medium or spread 500 μL on a selective plate
Colony PCR
Materials
• Materials for Taq PCR (except template plasmid DNA)
• Petri dish with transformed colonies
Procedure
• Prepare 25 μL reactions as in Taq PCR Protocol without template DNA
• With a sterile tip, under the flame, scrape part of a single colony and add to PCR tubes
• Mix by pipetting up and down or flicking the reactions
• Put tubes in thermocycler with cycling conditions as described in Taq PCR Protocol
Gibson Assembly - based on NEB Gibson Assembly Protocol
Materials
• DNA fragments
• 2X Gibson Assembly Mater Mix (NEB)
• 2X NEBuilder Positive Control (NEB)
• Deionized water
Procedure
• Set up following reactions on ice, adding Gibson Assembly Master Mix last:
| Component | 2 – 3 Fragments Assembly | 4 – 6 Fragments Assembly | Positive Control |
|---|---|---|---|
| Total Amount of Fragments | 0.02 – 0.5 pmols | 0.2 – 1 pmols | 10 μL |
| 2X Gibson Assembly Master Mix | 10 μL | 10 μL | 10 μL |
| Deionized water | to 20 μL | to 20 μL | 0 μL |
Optimized efficiency for 50 – 100 ng of vectors and 2 – 3 fold of excess inserts
• Incubate samples at 50°C for 15 minutes (2 – 3 fragments) or for 60 minutes (4 – 6 fragments)
• Store samples on ice or at -20°C until transformation
• Transform competent cells following the Transformation Protocol
Miniprep - with QIAprep Spin Miniprep Kit (QIAGEN)
Materials
• Bacterial overnight liquid cultures (1 - 5 mL)
• QIAprep Spin Miniprep Kit
Procedure
• Pellet 1 -5 mL bacterial culture by centrifugation at more than 8000 rpm for 3 minutes
• Resuspend pelleted bacterial cells in 250 μL P1 buffer and transfer to a microcentrifuge tube
• Add 250 μL P2 buffer and mix by inverting tube 4 – 6 times
• Add 350 μL N3 buffer and mix by inverting tube 4- 6 times
• Centrifuge for 10 min at 13000 rpm
• Apply supernatant to the QIAprep spin column by pipetting, centrifuge for 30 – 60 seconds and discard flow-through
• Wash the QIAprep spin column by adding 0.5 mL PB buffer, centrifuge for 30 – 60 seconds and discard flow-through
• Wash the QIAprep spin column by adding 0.75 mL PE buffer, centrifuge for 30 – 60 seconds and discard flow-through
• Centrifuge for 1 minute to remove residual wash buffer
• Elute DNA by placing QIAprep column in a clean 1.5 mL microcentrifuge tube and adding 50 μL EB buffer or water (or less for higher concentration). Let stand for 1 minute and centrifuge for 1 minute
Phusion PCR – based on NEB Phusion PCR Protocol
Materials
• 5X Phusion HF or GC Buffer
• dNTPs
• Forward and Reverse Primers
• Template plasmid DNA
• Phusion DNA polymerase
• Nuclease Free Water
Procedure
• Prepare following reaction in 0.5 mL PCR tubes on ice, adding polymerase last:
| Component | 20 μL reaction | 50 μL reaction |
|---|---|---|
| 5X Phusion HF or GC Buffer | 4 μL | 10 μL |
| 10 mM dNTPs | 0.4 μL | 1 μL |
| 10 mM Forward Primer | 1 μL | 2.5 μL |
| 10 mM Reverse Primer | 1 μL | 2.5 μL |
| Template plasmid DNA | 1 pg – 10 ng | 1 pg – 10 ng |
| Phusion DNA Polymerase | 0.2 μL | 0.5 μL |
| Nuclease Free Water | to 20 μL | to 50 μL |
Usually 100 pg – 1 ng of template DNA is sufficient
• Mix by pipetting up and down or flicking the reactions
• Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:
| Step | Temperature | Time | |
|---|---|---|---|
| Initial Denaturation | 98°C | 30 seconds | |
| 25 – 35 cycles | Denaturation | 98°C | 5 - 10 seconds |
| Annealing | 45 – 72°C | 10 – 30 seconds | |
| Extension | 72°C | 15 -30 seconds per kb | |
| Final Extension | 72°C | 5 -10 minutes | |
| Hold | 4°C |
Guidelines
To be completed
Taq PCR – based on NEB Taq PCR Protocol
Materials
• 10X Standard Taq Reaction Buffer
• dNTPs
• Forward and Reverse Primers
• Template plasmid DNA
• Taq DNA polymerase
• Nuclease Free Water
Procedure
• Prepare following reaction in 0.5 mL PCR tubes on ice, adding polymerase last:
| Component | 25 μL reaction | 50 μL reaction |
|---|---|---|
| 10X Standard Taq Reaction Buffer | 2.5 μL | 5 μL |
| 10 mM dNTPs | 0.5 μL | 1 μL |
| 10 mM Forward Primer | 0.5 μL | 1 μL |
| 10 mM Reverse Primer | 0.5 μL | 1 μL |
| Template plasmid DNA | 1 pg – 1 ng | 1 pg – 1 ng |
| Taq DNA Polymerase | 0.125 μL | 0.25 μL |
| Nuclease Free Water | to 25 μL | to 50 μL |
Usually 100 pg – 1 ng of template DNA is sufficient
• Mix by pipetting up and down or flicking the reactions
• Put tubes in thermocycler (with a pre-heated lid) with following cycling conditions:
| Step | Temperature | Time | |
|---|---|---|---|
| Initial Denaturation | 95°C | 30 seconds | |
| 25 – 35 cycles | Denaturation | 95°C | 15 – 30 seconds |
| Annealing | 45 – 68°C | 15 – 60 seconds | |
| Extension | 68°C | 1 minutes per kb | |
| Final Extension | 68°C | 5 minutes | |
| Hold | 4°C |
Guidelines
To be completed
PCR Product Purification - with QIAquick PCR Purification Kit (QIAGEN)
Materials
• PCR products
• QIAquick PCR Purification Kit
Procedure
• Add 5 volumes PB buffer to 1 volume of PCR product and mix
• Place QIAquick column in 2 ml collection tube
• Apply samples to QIAquick column and centrifuge for 30 – 60 seconds, discard flow-through
• Wash by adding 0.75 μL PE buffer to QIAquick column and centrifuge fo 30 – 60 seconds, discard flow-through
• Centrifuge QIAquick column for 1 minutes to remove residual wash buffer
• Elute DNA by adding 30 or 50 μL EB buffer or water to the center of the QIAquick column. Let stand for 1 minutes and centrifuge for 1 minute
PEG/LiAc Solution
Materials
• 50% PEG (Polyethylene glycol) prepared with sterile deionized water
• 10X TE buffer: 0.1 M Tris-Hcl, 10 mM EDTA, ph 7.5, autoclaved
• 10X LiAc: 1 M lithium acetate, pH 7.5 adjusted with dilute acetic acid, autoclaved
Procedure
• Prepare PEG/LiAc solution as follows:
| Stock concentration | Final concentration | Total quantity for 10 mL solution |
|---|---|---|
| 50% PEG | 40% PEG | 8 mL |
| 10X TE buffer | 1X TE buffer | 1 mL |
| 10X LiAc | 1X LiAc | 1 mL |
Sd Medium<7h1>
Materials
• Amino Acid Powder
• Yeast Nitrogen Base
• Ammonium Sulphate
• Adenine Sulphate
• Water
• NaOH
• Agar
• Glucose
Procedure
• Place stirrer bar in 2 L Erlenmeyer
• Add 2.6 g amino acid powder, 3.4 g yeast nitrogen base, 10 g ammonium sulphate, 1 g adenine sulphate and 950 mL water
• Adjust pH to 5.9 by adding a few drops of 10 M NaOH
• In an other Erlenmeyer, add 35 g agar and 900 mL water
• Autoclave both bottles
• Transfer the content of first bottle to the agar-containing bottle
• Cool to 55°C
• Add 100 ml 40% glucose and 16 ml of the required amino acids
• Pour plates
Transformation – based on NEB Transformation Protocol
Materials
• Competent cells
• DNA
• SOC medium (SOB + Glucose)
• Petri dish with appropriate antibiotic resistance
Procedure
• Thaw competent cells on ice
• Add 2 μL DNA to the competent cells, mix by pipetting up and down or flicking the tube 4 -5 times
• Place mixture on ice for 30 minutes
• Heat shock at 42°C for 30 seconds
• Transfer tubes to ice for 2 minutes
• Add 950 μL room-temperature SOC media
• Incubate at 37°C for 60 minutes with shaking
• Spread 100 μL cells onto selection plates (warm plates to 37°C prior to this step for increased efficiency)
• Incubate overnight at 37°C