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:

reagent	volume (in µL)
10x T4 DNA ligase buffer	2.5
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
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.
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.

Centrifuge the empty GeneJet purification column for an additional 1 min to completely remove residual wash buffer

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
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.
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.
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
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

Promega Wizard SV Gel and PCR CleanUp System

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 weight	mass to weigh (in mg) to obtain desired stock-solution	concentration in stock solution in mM
Alanine	89.09	182	4089
Arginine	174.20	202	2314
Asparagine-Monohydrate	150.14	282	3759
Aspartic acid	133.10	250	3752
Cysteine	121.16	149	2465
Glutamic acid	147.13	269	3655
Glutamine	146.15	183	2501
Glycine	75.07	158	4210
Histidine	155.15	255	3281
Isoleucine	131.18	247	3765
Leucine	131.18	167	2549
Lysine	146.19	175	2392
Methionine	149.21	186	2491
Phenylalanine	165.19	142	1716
Proline	115.13	224	3883
Serine	105.90	209	3953
Threonine	119.12	229	3853
Tryptophan	204.23	170	1661
Tyrosine	181.19	217	2396
Valine	117.15	152	2595

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 mixture	Concentration in mixture (without water and glacial acetic acid added)	concentration in final mixture
Alanine	13.6	146	13.56
Arginine	22.2	135	12.53
Asparagine-Monohydrate	13.6	134	12.44
Aspartic acid	13.6	134	12.44
Cysteine	22.2	143	13.28
Glutamic acid	13.6	130	12.07
Glutamine	22.2	145	13.46
Glycine	13.6	150	13.93
Histidine	13.6	117	10.86
Isoleucine	13.6	134	12.44
Leucine	22.2	148	13.74
Lysine	22.2	139	12.91
Methionine	22.2	145	13.46
Phenylalanine	34	153	14.21
Proline	13.6	138	12.81
Serine	13.6	141	13.09
Threonine	13.6	137	12.72
Tryptophan	34	148	13.74
Tyrosine	22.2	139	12.91
Valine	22.2	151	14.02
sum	381.6	2807	260.62

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!

Transfer culture into prechilled and weighted harvest tubes or falcons
Centrifugate: 5000x g, 4 °C, 15 min. While centrifugating, add DTT to S30 buffer to a final concentration of 2 mM
Discard supernatant and weigh pellets
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.
Centrifugate: 5000x g, 4 °C, 12 min
Discard supernatant
Repeat steps 4 to 6 two times
Centrifuge a last time at 5000x g, 4 °C, for 5 min and remove residual washing buffer by pipetting.
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.

Team:Bielefeld-CeBiTec/Protocols