Team:Kyoto/Protocol

div class="clear">

    Table

On this page, the ecolibrium team would like to share with you the protocols that we have been using over the summer. This library of protocols has been developed alongside our supervisors for the purpose of this study. Here you can find the exact methods we use to generate our data and results. We share them in the interest of reproducibility.

Materials:
Competent E.coli(Dh5alpha) LB broth
Ice
Selection plates

Methods:

  1. Thaw 10µL competent E. coli cells on ice for 10 minutes
  2. Add:
    • -2.5 µl DNA from a ligation reaction mix or
    • DNA of a known plasmid
  3. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  4. Place the mixture on ice for 15 minutes. Do not mix.
  5. Heat shock at exactly 42°C for exactly 45 seconds. Do not mix.
  6. Place on ice for 1-3 minutes. Do not mix.
  7. Pipette 100 µl of room temperature SOC or LB media into the mixture.
  8. Incubate at 37°C for 40 minutes.
  9. Mix the cells thoroughly by flicking the tube and inverting.
  10. Spread:
    • All of each transformation reaction onto a selection plate. If the reaction is too much　:
      1. Pellet cells at 8000rpm for 3 minutes
      2. Remove and dispense appropriate volume of supernatant
      3. Re-suspend cells by light vortexing
      4. Plate resuspended cells as above

Materials:
3 ml LB broth
3 μl antibiotic
Tips
culture tube

Methods:
Overnight cultures were prepared under sterile conditions using a clean bench

  1. Add 3 ml liquid LB media into 12 ml culture tubes
  2. Add 3 μl of appropriate antibiotic into the broth
  3. Using the tip, pick a single colony and inoculate the cultures by dipping the loop into the LB broth
  4. Seal the tubes and incubate overnight at 37°C shaking at 130-150 rpm

Materials: We used various PCR Polymerase, and we obeyed each protocols. We show our most frequent protocol here.
2x Q5 Mastermix
10 µM forward primer
10 µM reverse primer
PCR tube
Sterile water
Plasmid DNA

Methods:


For a 50 µL reaction

  1. In a PCR tube on ice, combine 1-10 ng of plasmid DNA, 2.50 µL of 10 µM forward primer, 2.50 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Q5 Mastermix, and sterile water up to 50 µL.
    Note: It is important to add Q5 Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity
  2. Gently mix the reaction
  3. If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
  4. Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling


Thermocycling
The PCR machine should be set to run the following steps:

Step Temperature (°C) Time
Initial denaturation 98 30 seconds
25-35 cycles 98 (denaturation)
45-72 (annealing) see Note 1
72 (extension)		 5-10 seconds
10-30 seconds
15-30 seconds per kb
Final extension 72 2-5 minutes
Hold 4 Indefinitely

Note 1: Use the NEB Tm calculator should be used to determine the annealing temperature when using Q5: http://tmcalculator.neb.com/#!/

Materials:
Sterile Water
2x EmeraldAmp MAX mastermix
E. coli colony
10 µM forward primer
10 µM reverse primer

Methods:

  1. Make mastermix of reaction.Add 2.5µL of EmeraldAmp MAX,0.05µL of 10µM forward primer, and 0.05µL of 10µM reverse primer to 2.4µL of water each reaction.
  2. Touch your objective colony with tip then touch the 5µL of reaction liquid in PCR tube.
  3. If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly


Thermocycling
The PCR machine should be set to run the following steps:

Step Temperature (°C) Time
Initial denaturation 98 30 seconds
25-35 cycles 98 (denaturation)
45-72 (annealing) see Note 1
68 (extension)		 5-10 seconds
10-30 seconds
15-30 seconds per kb
Final extension 72 5-10 minutes
Hold 4 Indefinitely

Note: If loading on a gel, the EmeraldAmp MAX mix contains loading dye, so don’t add anything else.

LB Broth
Materials:
10 g Tripton
5g Yeast Extract
5g NaCl
1L Purified Water

Methods:

  1. Add these reagents to 1L purified water
  2. Autoclave


LB Agar
Materials:
10 g Tripton
5g Yeast Extract
5g NaCl
15g Agar
1L Purified Water

Methods:

  1. Add these reagent to 1 L purified water
  2. Autoclave


Glycerol Stocks
Materials:
500µl glycerol (80%)
500µl overnight culture in LB

Methods:

  1. Add 500µl glycerol (80%) to 1.5ml eppendorf tube
  2. Add 500µl overnight culture in LB
  3. Store at -80°C

Materials:
Agarose Powder
TAE buffer
Gel mould
2-4µL EtBr Solution(10mg/ml)
Gel Tank
5-7 µL DNA ladder
DNA loading dye

Methods:

  1. Prepare 1% w/v solution of agarose powder in 1x TAE buffer (e.g. 1g agarose powder in 100 mL buffer) using a conical flask
  2. Heat the mixture until agarose is completely dissolved. Do not let the solution boil.
  3. Pour the solution into a gel mould
  4. Add 2-4 µL EtBr Solution (10mg/ml) to the solution. Make sure there are no bubbles in the solution.
  5. Allows the solution to set (approx 20-30 minutes)
  6. Transfer the agarose gel to a tank, remove the comb and apply:
    • 5-7 µL of the DNA ladder
    • DNA samples with the corresponding amount of DNA loading dye (10X)
  7. Run the gel for 25-35 minutes at 100V

Materials:
Microcentrifuge tube
Ice
2 µL 2X Ligationhigh
Vector Plasmid solution
Insert DNA solution

Methods:

  1. Calculate volumes of vector and insert DNA required using NEBioCalculator (required molar ratio of 1:3::vector:insert)
  2. Add vector plasmid, insert DNA, and 2x Ligationhigh to the microcentrifuge tube on ice (add T4 DNA ligase last)
  3. Incubate at 16℃ for 30 - 60 min for sticky ends or 2-16 hours for blunt ends

Materials:
Restriction Enzyme: NEB enzyme finder used to see which restriction enzymes are required
10X buffer: NEB double digest finder used to see which buffers are required for the particular restriction enzymes
Plasmid DNA
Sterile water

Methods:

Component Test digest
Double digestion (20 µL, for construct analysis) 		Assemble digest
Double digestion (20-50 µL, for gene assembly)
Sterile water to 20 µL to 50 µL
10X buffer 2 µL 2-5 µL
Plasmid DNA ~200 ng for test digest, (DNA mass is variable dependent on insert size.
Smallest digestion fragment mass should be > 50ng)		 1,000 -2,000 ng
Restriction enzyme 0.5 µL + 0.5 µL 1 µL + 1 µL
  1. Set up the reaction following the instruction in the table above , depending on whether test digest or assembly digest is being performed.
  2. Incubate digestion reaction at 37°C:
    1. 30 min for Test digest
    2. 2-3 hours or overnight for Assembly digest
  3. Perform heat deactivation at 80°C for 20 minutes, if not running on a gel at the end of incubation.

PCR purification was performed according to the FastGene™Gel/PCR Extraction Kit
Gel extraction was performed according to the FastGene™Gel/PCR Extraction Kit
Minipreps were carried out according to the FastGene™Plasmid Mini Kit