Team:Pasteur Paris/Protocols/CellBio

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Molecular Biology: general protocols

Here we present the basic molecular biology methods we used throughout the project to amplify our plasmids, linearize them and insert our sequences, retrieve them from bacteria and express proteins.

Agarose Gel Preparation

Bacterial Stock

Gel Extraction

Ligation

Liquid Culture

Midiprep for plasmid extraction

Miniprep for plasmid extraction

Transformation of E. coli BL21 (DE3) pLys

Transformation of E. coli DH5-alpha


Aim

Prepare an 8% agarose gel for the electrophoresis of DNA samples.


Materials

  • UltraPure™ Agarose (Invitrogen, 16500-100)
  • UltraPure™ 10X TAE Buffer (Invitrogen, 15558)
  • Gel Green Nucleic Acid Stain (Biotium, 41005)
  • Scale
  • Microwave
  • Spatula
  • Erlenmeyer (250 mL)
  • Measuring cylinder
  • PowerPac™ Basic (Bio-Rad, 1645050)
  • Mini-Sub® Cell GT Horizontal Electrophoresis System (Bio-Rad, 1704406)

Procedure


  1. Prepare 600 mL of TAE 1X by diluting 60 mL of 10X buffer in 540 mL of deionized water.
  2. Weigh 0,6 g of agarose on a scale.
  3. Place the agarose in an Erlenmeyer.
  4. Fill the Erlenmeyer with 75 mL of TAE 1X.
  5. Heat the Erlenmeyer for 2 min 30 s at 350W.
  6. Mix and place it again in the microwave for an additional minute.
  7. Let the mixture cool down a little bit and add 5 &Mu L of Gel Green.
  8. Pour the agarose in the horizontal electrophoresis system. Don’t forget to place the comb before!
  9. Let the gel cool down for 20-30 minutes before deposing the samples.



Aim

Stock bacterial culture at -80 °C.


Materials

  • Desired bacterial cultures on petri dish
  • Sterile LB media
  • Accurate antibiotics: Carbenicillin (50 mg/mL) or Chloramphenicol (25 mg/mL)
  • Glycerol 50%
  • Dry Ice
  • Falcon 15 mL and 50 mL
  • Erlenmeyer 125 mL
  • Sterile cryotube
  • Inoculation loop
  • Pipette p200 + p20 and associated cones
  • Plastic graduated pipette (25 mL)
  • Electric Pipetman

Procedure

In advance:

  • Prepare a stock solution of LB + desired antibiotic in 50 mL falcon tube depending on how many culture you would like to stock in glycerol.
  • Prepare a sterile stock solution of glycerol 50 %.
  1. In 15 ml sterile falcon, add 5 mL of LB media
  2. Vortex the stock solution of antibiotic and add 5 µL to the LB
  3. Using an inoculation loop, gently touch a colony of transformed bacteria from the petri dish, plastic side facing you.
  4. Immerse and dip the inoculation loop in the liquid media and stir.
  5. Place the liquid culture in the incubator at 37˚C and 180 rpm for 16 h.
  6. After 16 h, centrifuge the tubes 5 minutes at 3000 rpm.
  7. Discard supernatant.
  8. Resuspend the pellet in 5 mL of LB.
  9. Discard supernatant.
  10. Resuspend the pellet in 1 mL of fresh sterile LB medium + desired antibiotic
  11. In a 125 mL Erlenmeyer, add 1 mL of bacterial culture in 24 mL of LB + desired antibiotic.
  12. Incubate the culture at 37°C and 180 rpm.
  13. Measure the OD every hour for the first 3 h and then every 20 minutes.
  14. When the OD reaches 0.6 to 0.7, withdraw 5 mL of the bacterial liquid culture and add 5 mL of glycerol 50%.
  15. Vortex the tube.
  16. Aliquot the 10 mL into sterile cryotubes.
  17. Place into dry ice and freeze at -80°C.



Aim

Extract a specific DNA band from an agarose electrophoresis gel.


Materials

  • QIAquick Gel Extraction Kit (Qiagen, 28706)
  • Scale
  • Scalpel
  • Heating block
  • Water bath

Procedure


According to the QIAquick Gel Extraction Kit's manual

  1. Using a UV light, excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
  2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 &Mu L).
  3. Incubate at 50°C for 10 minutes (or until the gel slice has completely dissolved). To help dissolve the gel, mix by vortexing the tube every 2 to 3 minutes during the incubation.
  4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
  5. Add 1 gel volume of isopropanol to the sample and mix.
  6. To bind DNA, pipet the sample onto the QIAquick column and apply vacuum. After the sample has passed through the column, switch off the vacuum source.
  7. (Optional): Add 0.5 mL of Buffer QG to QIAquick column and apply vacuum.
  8. To wash the column, add 0.75 mL of Buffer PE to QIAquick column and apply vacuum.
  9. Transfer QIAquick column to a clean 1.5 mL microcentrifuge tube or to a provided 2ml collection tube. Centrifuge for 1 minutes at 13,000 rpm (~17,900 x g).
  10. Place QIAquick column in a clean 1.5 mL microcentrifuge tube.
  11. To elute DNA, add 50 &Mu L of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the centre of the QIAquick membrane and centrifuge the column for 1 minute at 13,000 rpm (~17,900 x g). Alternatively, for increased DNA concentration, add 30 &Mu L elution buffer, let stand for 1 min, and then centrifuge for 1 minute.



Aim

To perform the ligation of one or more inserts in a plasmid using the In-Fusion cloning kit.


Materials

  • Stellar Competent cells (Takara Clontech)
  • Linearized plasmid
  • Purified insert(s)
  • 5X In-Fusion HD Enzyme Premix (Takara Clontech)
  • Control plasmid pUC19
  • Control insert
  • Deionized water
  • Water bath at 50°C
  • 1.5 mL Eppendorf tubes
  • Heating block at 80°C
  • Dry ice

Procedure


  1. Set the mix between insert and linearized vector in molar ratio 2:1 and complete with distilled water to reach a reaction volume of 16 &Mu L. The optimal quantity of vector is 100-150 ng.
  2. Pre-heat vector and insert for 5 minutes at 80°C.
  3. Put on ice for 3 minutes.
  4. Add 4 µL 5X In-Fusion HD Enzyme Premix and let the cloning occur in a water bath at 50°C.
  5. Set on ice and proceed to transformation in Stellar competent cells.



Aim

Start a liquid culture of transformed bacteria to amplify a plasmid.


Materials

  • Petri Dish with LB agar media + antibiotics carbenicillin 50 &Mu g/mL or chloramphenicol 25 &Mu g/mL
  • Sterile LB culture medium
  • Antibiotics: Carbenicillin 50 mg/mL or Chloramphenicol 25 mg/mL
  • Shaking incubator at 37°C
  • Sterile 250 mL Erlenmeyer or 50 mL Falcon tube
  • Inoculation loop
  • Plastic graduated pipette (10 mL or 20 mL)
  • Electric Pipetman

Procedure


  1. Add 5 mL of sterile LB culture medium + appropriate antibiotics to a 15 mL falcon tube (or Erlenmeyer).
  2. Using an inoculation loop, touch a colony of transformed bacteria from the petri dish. Immerse the inoculation loop in the liquid medium and stir energetically.
  3. On a new petri dish, spread the rest of the bacterial colony in a zig-zag movement.
  4. Place the liquid culture in the incubator at 37°C for 14 hours at 180 rpm. Maintain the lids on top using tape but do not close the tubes. The liquid cultures are then good to use for further experiments.
  5. Place the petri dish in the incubator at 37°C for 14 hours and then store it at 4°C.



Aim

Retrieve and purify the amplified plasmids from a culture of transformed bacteria.


Materials

  • 25 mL of overnight liquid bacterial culture of a transformed colony
  • QIAfilter™ Plasmid Midi Kit (Qiagen, 12243)
  • Centrifuge
  • Falcon tube 50 mL
  • Electric pipetman

Procedure


According to the QIAfilter™ Plasmid Midi Kit's manual


  1. Harvest 25 mL of overnight bacterial culture by centrifugation at 6000 x g for 15 minutes at 4°C.
  2. Re-suspend the pelleted bacterial cells in 4 mL Buffer P1.
  3. Add 4 mL Buffer P2 and mix thoroughly by inverting the tube 4-6 times and incubate at room temperature for 5 minutes.
  4. Add 4 mL Buffer P3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  5. Pour the lysate into the barrel of the QIAfilter cartridge. Incubate at room temperature for 10 minutes, without inserting the plunger.
  6. Equilibrate a QIAgen-tip 100 by applying 4 mL Buffer QBT and allow the column to empty by gravity flow.
  7. Insert the plunger into the Cartridge and filter the cell lysate into the previously equilibrated QIAgen-tip. Let the cleared lysate enter the resin by gravity flow.
  8. Wash the QIAgen tip with 2 x 10 mL Buffer QC.



Aim

Retrieve and purify the amplified plasmids from a 5 mL culture of transformed bacteria.


Materials

  • 25 mL of overnight liquid bacterial culture of a transformed colony
  • QIAprep™ Spin Miniprep Kit (Qiagen, 27106)
  • Centrifuge
  • 1.5 mL Eppendorf tubes
  • Electric pipetman

Procedure


According to the QIAprep™ Spin Miniprep Kit's manual


  1. Pellet 5 mL of overnight bacterial culture by centrifugation at 6000 x g for 3 minutes at room temperature.
  2. Re-suspend the pelleted bacterial cells in 250 µL Buffer P1 and transfer to a 1.5 mL Eppendorf tubes.
  3. Add 250 &Mu L Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 minutes. If using LyseBlue reagent, the solution will turn blue.
  4. Add 350 &Mu L Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  5. Centrifuge for 10 minutes at 13,000 rpm in a table-top micro-centrifuge.
  6. Apply 800 &Mu L supernatant from step 5 to the QIAprep2.0 spin column by pipetting. Centrifuge for 30-60 s and discard the flow-through.
  7. Wash the QIAprep2.0 spin column by adding 0.75 mL Buffer PE. Centrifuge for 30-60 s and discard the flow-through. Transfer the QIAprep2.0 spin column to the collection tube.
  8. Centrifuge for 1 minute to remove residual wash buffer.
  9. Place the QIAprep2.0 column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 50 µL Buffer EB. Let stand for 1 minute, and centrifuge for 1 minute at 13,000 rpm.



Aim

Transform a plasmid in E. coli BL21 competent cells for protein expression.


Materials

  • LB agar plates covered with appropriate antibiotics
  • SOC Outgrowth Medium (NEB, B9020S)
  • BL21(DE3)pLysS Singles™ Competent Cells – Novagen (Merck, 70236)
  • Plasmids of interest
  • Shaking incubator at 37 °C
  • Stationary incubator at 37 °C
  • Water bath at 42 °C
  • Ice bucket filled with ice
  • Micro centrifuge tubes
  • Sterile spreading device

Procedure


  1. Remove agar plates from storage at 4°C and let them warm up to room temperature.
  2. Take competent cells out of -80°C and thaw on ice for approximately 5 minutes.
  3. Add 1 &Mu L of purified plasmid DNA to the tube of competent cells. Stir gently to mix and return to ice.
  4. Incubate the competent cell/DNA mixture on ice for 5 minutes.
  5. Heat shock each transformation tube into a 42°C water bath for 30 seconds.
  6. Put the tubes back on ice for 2 minutes.
  7. Add 250 &Mu L of SOC medium (without antibiotic) to the bacteria and grow at 37°C and 180 rpm for 60 minutes.
  8. Plate 250 &Mu L of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic.
  9. Incubate plates at 37°C overnight.



Aim

Transform a plasmid in E. Coli DH5-α competent cells for amplification and storage.


Materials

  • LB agar plates covered with appropriate antibiotics
  • SOC Outgrowth Medium (NEB, B9020S)
  • DH5-&Alpha Competent E. coli (High efficiency) (NEB, C2987 I)
  • Plasmids of interest
  • Shaking incubator at 37 °C
  • Stationary incubator at 37 °C
  • Water bath at 42 °C
  • Ice bucket filled with ice
  • Micro centrifuge tubes
  • Sterile spreading device

Procedure


  1. Remove agar plates from storage at 4°C and let them warm up to room temperature.
  2. Take competent cells out of -80°C and thaw on ice for approximately 25 minutes.
  3. Add 1 &Mu L of purified plasmid DNA to 100 &Mu L of competent cells. Mix by flicking the bottom of the tube and return to ice.
  4. Incubate the competent cell/DNA mixture on ice for 30 minutes.
  5. Heat shock each transformation tube into a 42°C water bath for 40 seconds.
  6. Put the tubes back on ice for 3 minutes.
  7. Add 600 &Mu L of SOC medium (without antibiotic) to the bacteria and grow at 37°C and 180 rpm for 40 minutes.
  8. Plate 250 &Mu L of the transformation onto a 10cm LB agar plate containing the appropriate antibiotic.
  9. Incubate plates at 37°C overnight.