Team:Bielefeld-CeBiTec/Protocols

iGEM Bielefeld 2015




Standard protocols

Blunt end ligation

  • For "intramolecular" ligation of a PCR product the 5'-ends are phosphorylated with T4 Polynucleotidekinase (PNK)
  • Set up the following reaction:
    • reagentvolume (in µL)
    10x T4 DNA ligase buffer2.5
    T4 PNK 1
    PEG 4000 50%2.5
    DNA (100 to 600 ng)x
    water to 25
  • Incubate at 37 °C, 30 min
  • Heat inactivate at 65 °C, 20 min
  • Add 1 µL T4 DNA ligase after reaction has cooled down to room temperature
  • Incubate at room temperature for at least 2 h, overnight also works.
  • Next step: Transformation via heat shock

Colony PCR

  • One Reaction mix contains
    • 5µL 5x GoTaq buffer (Promega)
    • 1 µL MgCl2 (25 mM stock)
    • 0.5 µL 10 mM dNTPs
    • 0.25 µL primer mix (prefix/suffix primers or sequencing primers) 100 mM
    • 17.625 µL ddH2O
    • 0.125 µL GoTaq polymerase (Promega)
    • 0.5 µL template
  • PCR program
    • Cell lysis and denaturation: 5 min, 95 °C
    • 30 cycles
      • Denaturation: 10 s, 95 °C
      • Hybridisation: 30 s, annealing temperature
      • Elongation: 60 s/kb of product, 72 °C
    • Final elongation: 5 min, 72 °C
  • Template alternatives
    • Pick a colony with sterile tip, elute in 100 µL ddH2O or buffer, store at 4 °C during PCR, plate if insert is of correct size
    • Pick colondy, streak at marked position on a new plate and solute remaining cells on the tip in the PCR tube with reaction mixture, cultivate if insert is of correct size
  • Gel electrophoresis for control of fragment size

DNA Purification

  • Thermo Scientific GeneJET Gel Extraction Kit

    • Note: all centrifugations should be carried out in a table-top microcentrifuge at >12000 x g/10.000-14.000 rpm
    • Preparation
      • Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slize into a pre-weighed 1.5 mL tube and weigh. Record the weigh of the gel slice. Note: If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.
      • Add 1:1 volume of Binding Buffer to the gel slice (volume:weigt)
      • Incubate the gel mixture at 50-60 °C for 10 min or until the gel slice is completely dissolved. Mix the tube by inverion every few minutes to faciliate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column.
      • Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
      • Optional: Use this step only when DNA fragment is <500 bp or >10 kb long.
        • If the DNA fragment is <500 bp, add 1:2 volume of 100% isopropanol to the solubilized gel solution. Mix thoroughly.
        • If the DNA fragment is >10 kb, add 1:2 volume of water to the solubilized gel solution. Mix thoroughly.
    • Binding of DNA
      • Transfer up to 800 µL of the solubilized gel solution (from step 3/4) to the GeneJET purification column. Centrifuge 1 min. Discard the flow-through and place the column back into the same collection tube.Note: If the total volume exceeds 800 µL, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow through after each spin. repeat until the entire volume has been applied to the column membrane. Do not exceed 1g of total agarose gel per column.
      • Add 100 µL of Binding Buffer to the GeneJet purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
    • Washing
      • Add 700 µL of Wash Buffer to the GeneJet purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube. (We altered the protocol to to wash steps a 350 µL optional)
      • Centrifuge the empty GeneJet purification column for an additional 1 min to completely remove residual wash buffer
    • Elution
      • Transfer the GeneJET purification column into a clean 1.5 mL microcentrifuge tube. Add 50 µL of Elution Buffer to the center of the purification column membrane. Centrifuge for 1 minute. Note: for low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µL does not significantly reduce the DNA yield. However, elution volumes less than 10 µL are not recommended. If DNA fragment is >10 kb, prewarm Elution Buffer to 65 °C before applying to column. If the elution volume is 10 µL and DNA amount is <5 µg, incubate column for 1 min at room temperature before centrifugation.(We altered the protocol to 3o µL Elution Buffer preheated to 60 °C and 1 min incubation before centrifuging for all sizes.)
      • Discard the GeneJet purification column and store the purified DNA at -20 °C.

  • Qiagen QIAquick Gel Extraction Kit (Centrifugation)

    • Note: All centrifugation steps are carried out at 17.900 g (13.000 rpm) in a conventional table-top microcentrifuge
    • Preparation
      • Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
      • Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume gel (100 mg gel - 100 µL). The maximum amount of gel per spin column is 400 mg. For >2 % agarose gels, add 6 volumes Buffer QG
      • Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2-3 minutes to help dissolve the gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow(similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.0, ad mix. The mixture turns yellow.
    • Binding of DNA
      • Add 1 volume isopropanol to the sample and mix.
      • Place a QIAquick spin column in a provided 2 mL collection tube. To bind DNA, apply the sample to the QIAquick column and centrifuge fo 1 min until all the samples have passed through the column. Discard the flow-through and place the QIAquick column back into the same tube. For sample volumes >800 µL, load and spin again.
    • Washing
      • If DNA will subsequently be used for sequencing, in vitro transcription or microinjection, add 500 µL Buffer QG to the QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube
      • To wash, add 750 µL Buffer PE to QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube. If the DNA will be used for salt-sensitive applications (sequencing, blut-ended-ligation) let the column stand 2-5 minutes after addition of Buffer PE. Centrifuge the QIAquick column in the provided 2 mL collection tube for 1 min to remove residual wash buffer
      • Place QIAquick column into a clean 1.5 mL microcentrifuge tube
    • Elution
      • To elute DNA, add 50 µL Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µL Buffer EB to the center of the QIAquick column, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time up to 4 min can increase the yield of purified DNA.
      • If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

  • Analytik Jena InnuPREP Gel Extraction Kit

    • Preparation
      • Excise the DNA fragment from the agarose gel with a sharp scalpel. Note: minimize the agarose gel slice. Check the weight. Do not use more than 300 mg gel slice for one spin filter.
      • Transfer the gel slice into a 1.5 mL or 2.0 mL reaction tube and add the 650 µL Gel Solubilizer.
      • Incubate for 10 min at 50°C until the agarose gel slice is completely dissolved.
    • Binding of DNA
      • Add 50 µL Binding Optimizer and mix the suspension by vortexing or piepetting some times up and down.
      • Apply the sample onto the spin filter (green) located in a 2.0 mL Receiver Tube. Close the cap and centrifuge at 10.000 x g (12.000 rpm) for 1 min. Discard the filtrate and re-use the Receiver Tube. Place the Spin Filter back into the 2.0 mL Receiver Tube.
    • Washing
      • Open the Spin Filter and add 700 µL Washing Solution LS, close the cap and centrifuge at 10.000 x g (12.000 rpm) for 1 min. Discard the filtrate and re-use the Receiver Tube. Place the Spin Filter back into the 2.0 mL Receiver Tube. Repeat the entire step.
      • Centrifuge at max speed for 2 min to remove all traces of ethanol. Discard the 2.0 mL Receiver Tube.
    • Elution
      • Place the Spin Filter into a 1.5 mL elution Tube. Carefully open the cap of the Spin Filter and add 30-50 µL Elution Buffer (optionally prewarmed to 50°C). Incubate at room temperature for 1 min. Centrifuge at 6.000 x g (8.000 rpm) for 1 min. A second elution step will increase the yield of extracted DNA. Note: The DNA can be eluted with a lower or a higher volume of Elution Buffer. (...) Store the extracted DNA at +4 °C. For long time storage placing at -20°C us recommended

  • Promega Wizard SV Gel and PCR CleanUp System

    • Gel Slice/PCR product preparation
      • Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5 mL microcentrifuge tube.
      • Add 10 µL of Membrane Binding solution per 10 mg of gel slice. Vortex and incubate at 50-65 °C until gel slice is copletely dissolved.
      • Alternative for PCR amplifications: Add an equal volume of Membran Binding Solution to the PCR amplification
    • Binding of DNA
      • Insert SV Minicolumn into Collection Tube.
      • Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 min.
      • Centrifuge at 16.000 x g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
    • Washing
      • Add 700 µL Membrane Wash Solution (ethanol added). Centrifuge at 16.000 x g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
      • Repeat previous Step with 500 µL Membrane Washing Solution. Centrifuge at 16.000 x g for 5 minutes.
      • Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.
    • Elution
      • Carefully transfer Minicolumn to a clean 1.5 mL microcentrifuge tube.
      • Add 50 µL of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16.000 x g for 1 minute.
      • Discard Minicolumn and store DNA at 4 °C or -20°C.

Gibson Assembly

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PCR

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Plasmid Isolation

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Standard BioBrick Assembly

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Transformation via Electroporation

  • Thaw 50 µl electrocompetent E. coli cells on ice, dilute with icecold 50 µl glycerine (10%) if necessary
  • Add 0.5-5 µl plasmid to 50 µl electrocompetent cells
  • Store cells on ice for 1 minute
  • Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ω
  • Transfer transformation reaction to 450 µl SOC-Medium and incubate 1 h at 37 °C
  • Plate on selective LB-Medium
  • Incubate over night at 37 °C

Transformation via Heat Shock

  • Thaw 100 µl chemo competent E. coli cells on ice
  • Add 0.5-5 µl plasmid to 100 µl chemocompetent cells
  • Store cells on ice for 10-30 min on ice
  • Heat shock for 90 seconds at 42 °C
  • Store reaction on ice for 60 seconds
  • Optional: Preheat SOC medium to 37 °C
  • Transfer reaction to 1 ml SOC medium and incubate at 37 °C for at least 1 hour
  • Centrifuge 3 minutes at 12000 rpm and plate on selective LB medium
  • Incubate at 37 °C over night

2015 InterLab protocols

Growing Cells for Measurement

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Plate Reader Measurement

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CFPS protocols

Amino acid preparation

  • According to protocol from Caschera and Noireaux 2015b. Weigh all aminoacids seperatly into microcentrifuge tubes. Add 500 µl of 5 M KOH to each amino acid. Solubilization is achieved via multiple inverting and, if necessary, vortexing. Especially tyrosine takes a while, and is a suspension rather than a solution. Stock solutions are afterwards stored at -20 °C. Note: Caschera and Noireaux say these stock solutions can only be stored a few weeks, however, we did not see a loss in performance after more than 3 months (16 weeks).

    • Molecular weightmass to weigh (in mg) to obtain desired stock-solution concentration in stock solution in mM
    Alanine 89.091824089
    Arginine 174.202022314
    Asparagine-Monohydrate 150.142823759
    Aspartic acid 133.102503752
    Cysteine 121.161492465
    Glutamic acid 147.132693655
    Glutamine 146.151832501
    Glycine 75.071584210
    Histidine 155.152553281
    Isoleucine 131.182473765
    Leucine 131.181672549
    Lysine 146.191752392
    Methionine 149.211862491
    Phenylalanine 165.191421716
    Proline 115.132243883
    Serine 105.902093953
    Threonine 119.122293853
    Tryptophan 204.231701661
    Tyrosine 181.192172396
    Valine 117.151522595

  • Combine stock solutions to an amino acid mixture like depicted below. Add water to 4 mL and 110 µL of glacial acetic acid to adjust pH to about 6.5. Aliquot (do not forget to mix properly!) and flash-freeze in liquid nitrogen, store at -80 °C.

    • Volume (in µL) for mixtureConcentration in mixture (without water and glacial acetic acid added) concentration in final mixture
    Alanine 13.614613.56
    Arginine 22.213512.53
    Asparagine-Monohydrate 13.613412.44
    Aspartic acid 13.613412.44
    Cysteine 22.214313.28
    Glutamic acid 13.613012.07
    Glutamine 22.214513.46
    Glycine 13.615013.93
    Histidine 13.611710.86
    Isoleucine 13.613412.44
    Leucine 22.214813.74
    Lysine 22.213912.91
    Methionine 22.214513.46
    Phenylalanine 3415314.21
    Proline 13.613812.81
    Serine 13.614113.09
    Threonine 13.613712.72
    Tryptophan 3414813.74
    Tyrosine 22.213912.91
    Valine 22.215114.02
    sum381.62807260.62

Cofactor premix

  • Stock solutions needed:
    • 2 M HEPES
    • 174 mM NAD
    • 33.9 mM folinic acid
    • 65 mM coenzyme A (CoA)
    • 50 mg/mL E. coli tRNA (Roche)
    • 200 mM putrescine
    • 1.5 M spermidine
  • Combine stock solutions to a create cofactor premix that is 20x final reaction concentration, depicted in the following list
    • 1 M HEPES
    • 6.6 mM NAD
    • 1.4 mM folinic acid
    • 5.4 mM coenzyme A (CoA)
    • 4 mg/mL E. coli tRNA (Roche)
    • 20 mM putrescine
    • 30 mM spermidine

Cell harvest

  • The following harvest protocol mainly orientates to the procedures in Sun et al. 2013 and Kwon and Jewett 2015.
  • Gather all materials needed: Harvest tubes and falcon tubes, washing buffer, DTT aliquot, liquid nitrogen, and a lot of ice. Start the harvest when E. coli culture reaches mid- to late exponential growth phase. For the E. coli we used in our experiments and cultivations, the mid- to late exponential growth phase was reached at an OD600 of 3-4 (see growth curves in notebook section).
  • harvest protocol - keep everything on ice between the steps!
    1. Transfer culture into prechilled and weighted harvest tubes or falcons
    2. Centrifugate: 5000x g, 4 °C, 15 min. While centrifugating, add DTT to S30 buffer to a final concentration of 2 mM
    3. Discard supernatant and weigh pellets
    4. Add about 10 mL of S30 washing buffer and resuspend cells by vortexing and vigourous shaking. For us, cycles of 15 s vortexing/shaking and 30 s resting on ice worked well.
    5. Centrifugate: 5000x g, 4 °C, 12 min
    6. Discard supernatant
    7. Repeat steps 4 to 6 two times
    8. Centrifuge a last time at 5000x g, 4 °C, for 5 min and remove residual washing buffer by pipetting.
    9. Flash freeze pellets in liquid nitrogen and store at -80 °C, unless you want to proceed with sonification directly.

Sonification

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Conduction of a CFPS reaction

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PRIA protocols

Plasmid Repressor Interaction Assay (PRIA)

  • After every step the reaction tube is centrifuged with 1000 g for 1 minute.
  • The supernatant after centrifugation and every wash step (starting from the step when the protein is added to the agarose) is stored for analyzing the samples for protein and DNA amounts.
  • As negative controls no protein to the agarose were added.
  • Steps:
    • 25 µL Ni-NTA agarose is put in a reaction tube. Then the sample is centrifuged.
    • The agarose is washed three times with Kpi buffer (Volume: 50 µL).
    • 250 pmol protein in 20 µL Kpi buffer is added and incubated for 30 min. Then the sample is centrifuged.
    • The agarose is incubated three times in Kpi Buffer (Volume: 50 µL).Then the sample is centrifuged.
    • 0.75 pmol plasmid (lacO) is mixed with 20 µL binding buffer (Incubation time: 15 min).
    • Unbound DNA is washed away three times with binding buffer (Volume: 50 µL, Incubation time: 15 min). Then the sample is centrifuged.
    • 3x elution with analytes in binding buffer (Volume: 50 µL, Incubation time: 15 min)
    • Imidazol solution (50 µl) is used to release proteins from the agarose.
    • Water (volume: 10 µL) is mixed with agarose.
    • The DNA amount of the supernatant after centrifugation is analyzed via gel electrophoresis.

Electrophoretic Mobility Shift Assay (EMSA)

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Immobilization of dsDNA on paper

  • Dissolve 10 mg p-Phenylenediisothiocyanate either in 30 ml pure ethanol or in 500 µL dried DMSO and add up to 30 ml with water. (the drying is essential, perform it with 4 Angström molecular sieves at least 24 h)
  • Whatman filter paper is activated for 1-2 days by orbital shaking in the PDITC solution.
  • Wash paper twice with DMSO or water respectively. Dry it for approximately 5 min
  • Generate an aminolabeled DNA fragement
    • The optimal concentration required for the immobilization is 20 µM. You can achieve this through hybridization of 2 Primers in 50 mM sodium phosphate buffer, pH 8.5. Boil the primers at 98 °C for a few minutes and afterwrds slowly decrease the temperature.
    • Alternatively, you can perform an conventional PCR with one of the primers aminolabeled. We recommend labeling of the other oligo with a fluorescent dye. This enables detection of the immobbilized DNA on Scanners like the Typhoon or Ettan Dige.
    • Pipett 1-0.1 µL onto the paper and incubate it over night in an humidification chamber (avoid direct contact of the liquid with the paper)
    • Block the paper with 50 °C warm blocking solution (0.1 M Tris, 50 mM ethanolamine, pH 9.0)
    • wash the paper three times for 2 min with water and dry it by an airstream. (If you wish to immobilze ssDNA, add an washing step with 50°C 4xSSC buffer, 30 min.)

Measurement of Fluorescence in Tecan

  • You can detect immobilized DNA und your fusion protein by measuring the fluorescence.
  • You need Cy3- and amino-labeled DNA with an operator site for immobilization, a repressor fused to a fluorescence protein and cellulose on a black 96-well plate.
    • Excitation for Cy3: 545 nm, Emission for Cy3: 590 nm
    • For example: Excitation wavelength for sfGFP: 480 nm, Emission wavelength for sfGFP: 515 nm
    • The gain needs to be adapted.
  • After every step the supernatant is transferred into another 96-well plate for measurement.
  • The plate with the cellulose is dried after having taken out the supernatant and measured again.
  • Cellulose on the plate is prepared in a black 96-well plate.
  • DNA is immobilized on cellulose.
  • Protein in Kpi buffer (Concentration: 20 µg/mL, Volume: 25 µL) is added to the wells with immobilized DNA (Incubation time: 15 min).
  • The wells are washed with binding buffer (Volume: 200 µL, incubation time: 10 min).
  • 100 µl analyte solution (e.g. 0.5 mM IPTG in binding buffer) is put in the well and shaken for 30 min.