Team:EPF Lausanne/Notebook/Protocols
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 (Based on NEB Taq PCR Protocol)
Materials
• 10X Standard Taq Reaction Buffer
• dNTPs
• Taq polymerase
• Forward and Reverse Primers
• Nuclease Free Water
• Petri dish with transformed colonies
Procedure
• Prepare following reaction in 0.5 mL PCR tubes on ice by making a Master Mix for all reactions and adding polymerase last:
| Component | 25 μL reaction | Final concentration |
|---|---|---|
| 10X Standard Taq Reaction Buffer | 2.5 μL | 1X |
| 10 mM dNTPs | 0.5 μL | 200 μM |
| 10 mM Forward Primer | 0.5 μL | 0.2 μM |
| 10 mM Reverse Primer | 0.5 μL | 0.2 μM |
| Taq DNA Polymerase | 0.125 μL | 0.625 units/25 μL PCR |
| Nuclease Free Water | to 25 μL |
• With a sterile tip, under the flame, scrape part of a single colony and add to PCR tube
• Mix by pipetting up and down or flicking the reactions
• Put tubes in thermocycler 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 - 60seconds | |
| Extension | 68°C | 1 minutes per kb | |
| Final Extension | 68°C | 5 minutes | |
| Hold | 4°C |
Gibson Assembly
Materials
• DNA fragments
• 2X Gibson Assembly Mater Mix (NEB)
• 2X NEBuiler 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
Section 4-1
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Section 4-2
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