Difference between revisions of "Team:Bielefeld-CeBiTec/Protocols"

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                     <p>...</p>
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                     <p>
 +
        <ul>
 +
            <li>You can detect immobilized DNA und your fusionprotein by measuring the fluorescence. </li>
 +
            <li>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.</li>
 +
            <ul>
 +
                <li>Excitation for Cy3: 545 nm, Emission for Cy3: 590 nm</li>
 +
            <li>For example: Excitation for sfGFP: 480 nm, Emission for sfGFP: 515 nm</li>
 +
            <li>The gain needs to be adapted.</li>
 +
            </ul>
 +
            <li>After every step the supernatant is transferred into another 96-well plate for measurement. </li>
 +
            <li>The plate with the cellulose is dried after having taken out the supernatant and measured again. </li>
 +
        </ul>
 +
        <ul>
 +
            <li>Cellulose on the plate is prepared in a black 96-well plate.</li>
 +
            <li>DNA is immobilized on cellulose.</li>
 +
            <li>Protein in Kpi buffer (Concentration: 20 µg/mL, Volume: 25 µL) is added to the well (Incubation time: 15 min).</li>
 +
            <li>Binding buffer is used for washing (Volume: 200 µL, incubation time: 10 min).</li>
 +
            <li>100 µl analyte solution (e.g. 0.5 mM IPTG in binding buffer) was put in the well and shaken for 30 min. </li>
 +
        </ul>
 +
</p>
 
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Revision as of 23:07, 20 August 2015

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

...

DNA Purification

...

Gibson Assembly

...

PCR

...

Plasmid Isolation

...

Standard BioBrick Assembly

...

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

...

Plate Reader Measurement

...

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: According to Caschera and Noireaux, these stock solutions can only be stored a few 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 everthing 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.

PRIA protocols

Plasmid Repressor Interaction Assay (PRIA)

  • After every step the reaction vessel is centrifuged with 1000 g for 1 minute.
  • The supernatant after centrifugation and 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 mo 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 25 µL Kpi buffer.
    • 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: 25 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 25 µL binding buffer (Incubation time: 15 min). Then the sample is centrifuged.
    • 3x elution with analytes in binding buffer (Volume: 25 µl, Incubation time: 15 min)
    • Imidazol solution (25 µl) is used to release proteins from the agarose.
    • The DNA amount of the supernatant after centrifugation is analyzed via gel electrophoresis.

Electrophoretic Mobility Shift Assay (EMSA)

...

Immobilization of dsDNA on paper

...

Measurement of Fluorescence in Tecan

  • You can detect immobilized DNA und your fusionprotein 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 for sfGFP: 480 nm, Emission 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 well (Incubation time: 15 min).
  • Binding buffer is used for washing (Volume: 200 µL, incubation time: 10 min).
  • 100 µl analyte solution (e.g. 0.5 mM IPTG in binding buffer) was put in the well and shaken for 30 min.