Team:Nantes/Experiments

Protocols & Experiments Protocols & Experiments

PCR

Material :
  • P10, P100
  • Tubes PCR (O.2 mL)
  • Thermocycler
  • Sterile water
  • 5x PHUSION HF
  • DNTPs (10uM)
  • Forward Primer (10uM)
  • Primer Reverse (10uM)
  • Diluted DNA (50mg/uL)
  • PHUSION DNA Pol (2U/uL)
Protocol :
  • Add in a PCR tube (total volume = 50uL) :
    • 32.5 uL of dilluted water
    • 10 uL 5x PHUSION HF
    • 1 uL of dNTP
    • 2.5 uL of Forward Primer
    • 2.5 uL of Reverse Primer
    • 1 uL diluted DNA
    • 0.5 uL PHUSION DNA Pol
  • Place the tube in the thermocycler and start the programm associated with the DNA used as a matrix
  • Store the result at -20°C (-4°F)

Gibson

Goal: Assemble the different DNA parts recuperated by PCR

Material :
  • PCR tube
  • Purified DNA
  • Pipette p10
  • Hot plate (50°C/122°F)
  • Gibson reactant
  • Vortex
  • RNase-free water
Protocol :
  • Deposit the adapted volumes of purified DNA in a PCR tube
  • Vortex quickly
  • Add 5uL of the Gibson reactant
  • Mix the solution by pipetting back and forth
  • Incubate at 50°C for 1h
  • Store the tube at -20°C (-4°F)

PCR Quick Change

Protocol :
  • Add into a 50uL PCR tube
  • 36uL of distilled water
  • 10uL of 5x reaction buffer
  • 1uL (we want to have 100 ng of DNA in a total volume of 50uL) of plasmid pSRL from Guy
  • 1uL (125 ng) of primer #1 (forward Quick Change)
  • 1uL (125ng) of primer #2 (reverse Quick Change)
  • 1 uL of dNTP
  • Just before the PCR add 1uL of pHusion
  • Program x20
    • Temparature Times
    95°C 30 sec
    95°C 30sec
    55°C 1min
    72°C 6min
    72°C 10min
    14°C infinity
  • Add 1uL of DPN1 incubate 1h at 37°C

Incubate of the PCR Quick Change with DPN1

Goal : This enzyme degrades the DNA matrix

Protocol :
  • Add 1 uL of DPN1 (in cold conditions!! Because of the presence of enzyme)
  • Incubate at 37°C for 1h in a PCR machine
  • Inactivate the enzyme at 80°C for a few minutes

Electrophoresis

Material :
  • pipettes p100, p20, p10
  • Load buffer
  • Size marker
  • Electrophoresis machine
  • PCR results
  • Centrifugation tubes
  • Electrophoresis gel
Protocol :
  • The loading buffer needs to be at a concentration of 1x in samples of 50uL : add 10uL to 50uL of PCR result
  • Preparation of 20 uL of size markers with loading buffer :
    • 1 uL of size markers x 5
    • 1 uL of loading buffer x 5
    • 4 uL of water x 5
  • Deposit 6 uL of the marker+loading buffer solution into 1 gel pit
  • Deposit 60 uL of each sample into 2 pits (30 uL in each)
  • Once the migration is finished, observe the results with UV rays. Be very cautious to respect all security guidelines. Minimise the DNA’s exposition to UV rays because the longer the DNA is exposed the higher the risk that it will be distorted
  • Cut out the pieces of gel that contain the DNA that migrated as wanted and store each piece in a centrifugation tube at -20°C (-4°F)

Digestion

Goal : Recuperate pieces of certain plasmids

Material :
  • H2O
  • NE Buffer
  • EcoR1 HF
  • Pst1
  • Spe1
  • Xba1
Protocol:
  • Vector : Add in the same tube (total volume = 50uL) :
    • 5 uL NE Buffer 2.1
    • X uL plasmid (1ug) (1000ng/50uL = 20ng/uL so : (20 x 50)/“concentration Nanodrop obtained from the mini prep” = XuL)
    • 1 uL of Eco RI HF
    • 1 uL of Pst1
    • Complete to 50uL with distilled water
  • Insert :
    • 1) Digestion EcoR1 HF / Spe 1
      • 5uL of NE buffer 2.1
      • X uL of Insert (500 mg) (500ng/50uL = 10ng/uL so 10x50/“Nanodrop concentration obtained Miniprep” = X uL)
      • 1 uL of Eco R1 HF
      • 1 uL Spe1
      • Complete to 50uL with distilled wate
    • Digestion Xba1/Pst1
      • 5uL of NE Buffer
      • (Same formula as before) XuL of insert (500mg)
      • 1 uL of Xba1
      • 1 uL of Pst1
      • Complete to 50 uL of H2O
        • Enzyme/Insert pLAC pARA pSRL pRIB
        EoR1-Spe1 x x x
        Xba1-Pst1 x x x
      • Incubate reaction at 37°C for 30 min
      • Inactivate 80°C for 20 minutes

Spectrofluorometry

Goal : Measure the Optical Density and the fluorescence of transformed bacteria in different mediums

Material :
  • Tecan
  • Bacterial Pre-Culture in
  • Pipette p100
  • Black plates with 96 pits (to measure fluorescence)
  • Transparent plates with 96 pits (to measure Optical Density)
  • Bunsen Burner
Protocol:
  • Deposit 143 uL of appropriate sugar medium, 7 uL of appropriate bacterial preculture and 50 uL of mineral oil into each plate
  • Measure the OD and fluorescence with the TECAN

Mini-preps (Kit EZNA -> Plasmid DNA mini kit I Quick Guide) provided by Omega

Material :
  • Centrifuge
  • Heating plate
  • Steril water
  • Reactants from the kit
  • P100 + cones
  • Nanodrop
Protocol :
  • From a bacterial pre-culture in 5 mL LB
  • Centrifugate at 7850 rpm for 2min30sec at room temperature 21°C
  • Eliminate the supernatant by turning the tube upside down in a biological liquid trash
  • Add 250 uL of the solution I and resuspend the pellet with the pipette
  • Transfer into an eppendorf tube of 2 mL
  • Lysis step : Add 250 uL of the solution II and mix cautiously by turning the tube upside down 1 or 2 times. Careful the bacteria are very fragile at this point
  • Wait around 1 minute until the solution becomes slimy. Do not go over 2 minutes!
  • Add 350 uL of the solution III and mix by turning the tube upside down and back until « white flakes » appear. Centrifugate at 11180 rpm during 15 minutes
  • Insert the column in the collector tube.*
  • Deposit the supernatant from the centrifugation in the column (Very sensitive step, be very careful)
  • Centrifugate for 1 min at maximum intensity
  • Add 500 uL of HBC buffer
  • Centrifugate at 11 180 rpm for 1 minute then eliminate the supernatant
  • Add 700 uL of DNA wash buffer
  • Centrifugate at 11 180 rpm for 1 minute then eliminate the supernatant
  • Add 700 uL of DNA was buffer
  • Centrifugate at 11 180 rpm for 1 minute then eliminate the supernatant
  • Centrifugate the open tubes at 11 180 rpm for 2 minutes (this allows the column to dry
  • Deposit the column in an eppendorf tube of 1.5 mL and let dry for 10 min (open tube)
  • Add 50 uL of hot sterile water and leave it to sit at room temperature for 1 minute
  • Centrifugate at 11 180 rpm for 1 minute
  • Measure the concentration and purity of the DNA with the Nanodrop
  • Store DNA at -20°C

Competence with TSS

Make bacteria competent, meaning to weaken their cell wall to facilitate the entry of plasmids inside the bacteria. This method is preferred on bacteria that were stocked beforehand

Material :
  • Spectrophotometer
Protocol :
  • Seed 25 mL of LB with 500 uL of pre-culture
  • Incubate at 37°C in an agitator. Monitor the growth by measuring the Optical Density at 550 nm. Stop the culture when the OD is between 0.45 and 0.5
  • Place the eppendorf tubes on ice for 10 minutes. Make aliquots of the culture of 1 mL in the tubes
  • Incubate the DNA/competent bacteria mix on ice for 30 minute
  • Centrifugate at 4000 G for 4 to 5 minutes at 4°C
  • Eliminate the supernatant in the biological liquid trash
  • Delicately resuspend (without agitating or pipetting) the pellets in 100 uL do cold TSS at 4°C
  • Store the tubes at -80°C

Test competence with transformation

Material :
  • Petri Dishes containing LB agar and antibiotic
  • Plasmid used to transform (pET11A)
  • Hot plate
  • Crushed ice
  • LB
  • Centrifuge
  • Agitator/Incubator
Protocol :
  • Put 1 uL of plasmid « pET11A » (90 ng of DNA) in an eppendorf tube containing the « competent » bacteria to be tested
  • Incubate for 30 minutes on ice
  • Thermal shock : incubate 45 seconds at 42°C then on ice for 2 minutes
  • Add 800 uL of LB
  • Incubate for 1 hour under agitation at 100 rpm
  • Centrifugate 5 minutes at 3000 G
  • Remove the supernatant and resuspend the pellet with 100 uL of this supernatant
  • Spread 100 uL of this solution on a Petri dish containing LB agar and the associated antibiotic
  • Incubate in oven at 37°C overnight
  • See if their are any colonies the next day

Competence with TB+

Make bacteria competent, meaning to weaken their cell wall to facilitate the entry of plasmids inside the bacteria. This method is preferred for bacteria that have been freshly grown

Material :
  • In cold chamber
  • Bacterial pre-culture
  • LB
  • Spectrophotometer
  • Sterile eppendorf tube of 1.5 mL
  • 2 falcon tubes of 50mL
  • Crushed ice
  • Pipette de 25mL and 50mL
  • Pipette p1000
Protocol :
  • From a bacterial preculture in 10mL of LB with antibiotics
  • Dilute the preculture 1/100
  • Incubate at 37°C with agitation
  • Follow the Optical Density at 550nm (blank = 650 uL of LB with antibiotics) Careful! Stop the culture when the OD is between 0.45 and 0.5 to have the right amount of bacteria
  • In the cold chamber
    • Separate the previous pre-culture in 2 eppendorf tubes of 50 mL (cold)
    • Incubate 10 minutes on ice
    • Centrifugation 10 to 15 minutes at 2500 rpm at 4°C
    • Eliminate the supernatant
    • Cautiously resuspend the pellet in 2mL (no vortex or aspiration/respiration with the pipette)
    • Mix carefully
    • Incubate 10 min on ice
    • Reassemble both tubes in 1 tube
    • Add DMSO at 7%
    • Mix carefully
    • Make aliquots of 100 uL of the competent bacteria (in eppendorf tubes of 1.5mL)
    • Store at -80°C

Bacterial fixation with PFA

Goal : Immediate stop bacterial growth at a precise moment

Material :
  • Incubator/Agitator
  • Bacterial pre-culture
  • Spectrophotometer
  • Centrifuge
  • Eppendorf tubes
  • Pipettes P1000, P200, P10
  • PFA at 4%
  • PBS (1x)
  • Crushed ice
Protocol :
  • From the bacterial pre-culture take 1 mL and measure the Optical Density at 550nm
  • From this same preculture take 1mL and place in an eppendorf tube (Triplicate samples) :
    • Centrifugate at 10000 G for 5 minutes
    • Eliminate the supernatant
    • Add 100 uL of PFA at 4%
    • Incubate 10 to 15 minutes on ice
    • Centrifugate at 10000 G for 5 minutes
    • Eliminate the supernatant (in a toxic PFA trash)
    • Add 100 uL of PBS
    • Centrifuge at 10000G for 5 minutes
    • Eliminate the supernatant
    • Add 100 uL of PBS
    • Centrifugate at 10000G for 5 minutes
    • Eliminate the supernatant
    • Add 400 uL of PBS
    • Store at 4°C

Bacterial transformation

Goal: Genetically modify bacteria by adding one or many genes

Material :
  • Agitator/Incubator at 37°C
  • Water bath at 42°C
  • Crushed Ice
  • Centrifugation tubes or 1.5mL tubes
  • Autoclave
  • LB medium
  • Petri dish containing LB agar and antibiotic (Ampicillin or Kanamycin depending on the plasmid used for the transformation)
  • Competent bacteria (XL1 Blue or K12)
  • Plasmid (We designed and received from the Integrated DNA Technologies)
Protocol :
  • Take the competent bacteria out of the freezer at -80°C and let thaw on ice for 20 to 30 minutes
  • Take the Petri-dishes out of the fridge at 4°C and let them warm up at room temperature
  • Dilute the DNA solutions in sterile water to obtain a concentration of 10ng/uL. Take 1 uL of this dilution and add it to 50uL of competent bacteria in a centrifugation tube. Mix delicately by tapping the bottom of the tube a few times
  • Incubate the DNA/competent bacteria mix on ice for 30 minute
  • Do a heat shock of each transformation tube by putting ½ or ⅔ of the tube in the water bath at 42°C for 30 to 60 sec
  • Put the tubes back on ice for 2min
  • Add 500 uL of LB (without antibiotics) to the bacteria and let grow 45 min in an agitator at 37°C . Place the tubes in a horizontal position
  • Pour 50uL of the transformation on a Petri dish containing the antibiotic associated with the plasmid used during the transformation
  • Incubate the Petri dishes at 37°C overnight

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