• For "intramolecular" ligation of a PCR product the 5'-ends are phosphorylated with T4 Polynucleotidekinase (PNK)
• Set up the following reaction:

reagent	volume [ µL]
10x T4 DNA ligase buffer	2.5
T4 PNK	1
PEG 4000 50% (w/v)	2.5
DNA (100 to 600 ng)	x
ddH2O	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

• One Reaction mix contains
• 5 µL 5x GoTaq buffer (Promega)
• 1 µL MgCl₂ (25 mM stock)
• 0.5 µL dNTPs (10 mM each)
• 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 colony, 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

• Reaction mix
• 30 µL purified vector DNA
• 4 µL 10x SAP buffer
• 2 µL SAP
• 4 µL H₂O
• Protocol
• Incubate for 30 min at 37 °C
• Inactivate for 15 min at 65 °C

• Generate the complenentary sequence overlaps by PCR using the Phusion DNA-polymerase. If necessary add 5 M Betain in the reaction mix by reducing the amount of H₂O to decrease the number of false PCR products.
• Seperate the PCR product of interest by gel electrophoresis, extract the products via Gel Extraction.

• Preparing assembly mixture
• 320 µL 5x isothermal reaction buffer
• 0.64 µL of 10 U ml^-1 T5 exonuclease (for DNA molecules overlapping by greater than 150 bp add 3.2 µL of 10 U mL^-1 T5 exonuclease)
• 20 µL of 2 U mL^-1 Phusion DNA polymerase
• 160 µL of 40 U mL^-1 taq DNA ligase
• add ddH₂O up to a final volume of 1.2 mL
• Aliquote 15 µL of the assembly mixture and store at -20 °C
• That 15 µL assembly mixture on ice until use
• Add 5 µL of the purified DNA molecules in equimolar amounts (between 10 and 100 ng of each fragment).
• Incubate the mixture at 50 °C for 15 to 60 min with 60 min being optimal.

• Protocol (Macherey-Nagel Purification of His-tag proteins Kit) Protino® Ni-TED 1000 Packed Columns Kit
• Preparation of cleared lysates under native conditions
• That the cell pellet from an E. coli expression culture on ice (if frozen). Resuspend 1 g of pelleted, wet cells in 2-5 mL LEW buffer, pipette up and down, or use stirring until complete resuspension without visible cell aggregates. Perform this step on ice
• Add lysozyme to a final concentration of 1 mg/mL. Stir the solution on ice for 30 min
• Sonicate the suspension on ice according to the instructions provided by the manufacturer (e.g. use 10 x 15 s bursts with a 15 s cooling period between each burst). Carefully check samples´ appearance after sonication. If the lysate is still viscous from incomplete fragmentation of DNA, add 5 µg/mL DNase I and stir on ice for 15 min.
• Centrifuge the crude lysate at 10.000 x g for 30 min at 4 °C to remove cellular debris. Carefully transfer the supernatant to a clean tube without disturbing the pellet. If the supernatant is not clear, centrifuge a second time or filter through a 0.45 µm membrane (e.g. cellulose acetate) to avoid clogging of the IMAC column with insoluble material. Store supernatant on ice.
• Purification protocol
• Column equilibration: Equilibrate Protino Ni-TED/IDA packed columns with 1 x LEW Buffer. Allow the column to drain by gravity (2.0 mL). Protino Ni-TED/IDA1000 packed columns are designed to fit into most 15 mL conical centrifuge tubes for convenient fraction collection
• Binding: Add the cleared lysate to the pre-equilibrated column and allow the column to drain by gravity
• Washing: Wash the column with 1 x LEW Buffer. Allow the column to drain by gravity (2 x 2.0 mL)
• Elution: Wlute the polyhistidine-tagged protein in a new collection tube by adding 1 x Elution Buffer. Allow the column to drain by gravity (3 x 1.5 mL). Use protein assay and/or SDS-PAGE analysis to determine which fraction(s) contain(s) the majority of the polyhistidine-tagged protein.

• Modified from Silver Lab
• Minimum size of the BioBricks for this methods is 150 bp, whereas it should be at least 500 bp bigger or smaller than the used backbone. The BioBricks, which complies with these conditions, is used as the insert and is assembled into the prefix or suffix of the other BioBrick called vector. The methods differ in prefix and suffix insertion.
• Suffix insertion
• Digestion of inserted Biobrick
• Acquire > 700 ng DNA (10 µL volume)
• Add 1 µL 10 x NEBuffer
• Add 0.5 µL XbaI
• Add 0.5 µL PstI
• Digest 1 h at 37 °C
• Inactivate 20 min at 80 °C
• Clean up via gel electrophoresis, cut and extract from gel
• Digestion of the vector
• Acquire about 700 ng DNA (10 µL volume)
• Add 1 µL 10 x NEBuffer 2.1
• Add 0.5 µL SpeI
• Add 0.5 µL PstI
• Digest 1 h at 37 °C
• Inactivate 20 min at 80 °C
• Add 1 µL Antarctic Phosphatase (AP) and 1.2 µL 10 x AP reaction buffer
• Incubate 1 h at 37°C
• Clean up with PCR clean up kit
• Ligation
• Mix 50-200 ng vector with 3-10 fold molar access of insert
• Add 2 µL T4-Ligase-Buffer, 1 µL T4-DNA-Ligase
• Incubate for a minimum 20-30 min at room temperature (we usually ligate over night)
• Inactivate 5 min at 70 °C
• Store at -20 °C or transform
• Prefix insertion
• Follow protocol of Suffix insertion, replace the restriction enzymes as following:
• Digest insert with EcoRI and SpeI
• Digest vector with EcoRI and XbaI

• 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

• 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

• For 2 resolving gel (14 %):
• 4.7 mL Bisacrylamid/Acrylamid (0.8 %; , 30 %;, at the ratio of 37.5:1)
• 2.7 mL H₂O
• 2.5 mL 0.5 M TRIS-HCl, pH 6.8
• 0.1 mL 10% SDS

• 1.3 mL Bisacrylamid/Acrylamid (0.8 %; , 30 %;, at the ratio of 37.5:1)
• 6.1 mL H₂O
• 2.5 mL 0.5 M TRIS-HCl, pH 6.8
• 0.1 mL 10% SDS
• 50 µL 10 % ammonium persulfate
• 5 µL TEMED

• Remove sealing and store the gel wrapped in moistened paper towel at 4 °C.

• Mix your protein mixture 3:1 with PBJR buffer (15 µL protein solution + 5 µL PBJR buffer)
• Heat for 5 minutes at 95 °C

• Remove sealing, put the polymerized gel into gel box and pour 1x SDS-PAGE running buffer into the negative and positive electrode chamber.
• Remove comp without destroying the gel pockets.
• Pipet the SDS running buffer in the gel pockets up and down for flushing the gel pockets.
• Pipet slowly 20 µL of the sample into the gel pockets.
• Make sure to include at least one lane with molecular weight standards (PageRuler Prestained Protein Ladder™ (Fa. Fermentas)) to determinate the molecular weight of the sample.
• Connect the power lead and run the gel with 180 V until the distance of the lowest molecular weight standard lane to the gel end is down to 0.5 cm.

• After finishing the SDS-PAGE remove gel from gel casting form and transfer it into a box.
• Add 100 mL of the Colloidal Coomaassie Brilliant Blue staining solution to your polyacrylamid gel.
• Incubate the gel in the solution at room temperature until the protein bands got an intensive blue color. Shake the gel continuously during incubation.
• Remove the staining solution.
• Wash the gel with destaining buffer.
• Incubate the gel in H₂O (2-6 h) for bleaching the background. Shake the gel continuously during incubation. If necessary replace the colored water with new one.

• Be careful that skin scales or hair do not contaminate your sample. So wear hand gloves and tie up your hair.
• Reaction tubes have to be cleaned with 60 % (v/v) CH₃CN and 0.1 % (v/v) TFA. Afterwards the solution has to be removed completely followed by evaporation of the tubes under a fume hood. Alternatively, microtiter plates from Greiner® (REF 650161) can be used without washing. If you work with reaction tubes from Eppendorf, you do not need the wash step either.
• Cut out the protein lanes of a Coomassie-stained SDS-PAGE using a clean scalpel. Gel parts are transferred to the washed reaction tubes. If necessary, cut the parts to smaller slices
• Gel slices should be washed two times. Therefore add 200 µL 30 % (v/v) acetonitrile in 0.1 M ammonium hydrogen carbonate each time and shake lightly for 20 minutes till the gel slices are destained. Remove supernatant and discard to special waste
• Dry gel slices at least 30 minutes in a Speedvac.
• Rehydrate gel slices in 15 µL trypsin solution followed by short centrifugation.
• Trypsin-solution: 1 µL trypsin + 14 µL 10 mM NH₄HCO₃
• For this solution solubilize lyophilized trypsin in 200 µl of provided buffer and activate Trypsin for 15 minutes at 30 °C. For further use it can be stored at -20 °C.
• Gel slices have to be incubated 30 minutes at room temperature, followed by incubation at 37 °C overnight
• Dry gel slices at least 60 minutes in a Speedvac.
• According to the size of the gel slice, add 5 - 20 µL 50 % (v/v) ACN / 0.1 % (v/v) TFA
• Samples can be used for MALDI measurement or stored at -20 °C

• Spot 0.5 - 1 µL of sample aliquot
• Add 1 µL HCCA matrix solution to the spotted sample aliquots. Pipet up and down approximately five times to obtain a sufficient mixing. Be careful not to contact the AnchorChip. Note: Most of the sample solvent needs to be gone in order to achieve a sufficiently low water content. When the matrix solution is added to the previously spotted sample aliquot at a too high water content in the mixture, it will result in undesired crystallization of the matrix outside the anchor spot area.
• Dry the prepared spots at room temperature
• Spot external calibrants on the adjacent calibrant spot positions. Use the calibrant stock solution (Bruker’s “Peptide Calibration Standard II”, Part number #222570), add 125 µL of 0.1 % TFA (v/v) in 30 % ACN to the vial. Vortex and sonicate the vial.
• Mix the calibrant stock solution in a 1:200 ratio with HCCA matrix and deposit 1 µL of the mixture onto the calibrant spots.
• Analyze samples in ultrafleXtreme by Bruker Daltonics.

Preparation

• Prepare overnight cultures of the strains you want to test with the BioLector.

Materials

• Growth media (LB)
• Overnight cultures of tested strains
• Gas Permeable Seal
• 48-well plates

Procedure

• Measure OD of the overnight cultures.
• Centrifuge the overnight cultures and discard the supernatant.
• Add LB-medium till an o.D. of 0,1.
• Add 1 mL bacteria suspension in every well of the 48-wellplate.
• Stick the Adhesive Gas Permeable Seal to the plate and make sure it is stuck on tightly.
• Apply the Adhesive Seal for Evaporation Reduction on top of the Gas Permeable Seal.
• Put the BioLector plate into the BioLector in the specified slot.
• Set the temperature and moisture to be held during the measurement using the Start Assistant in the BioLector.determine the used filters for biomass measurement 620nm and for fluorescence measurement 486nm (ex) and 510 (em).
• Run the BioLector for circa 41 hours.
• Use the following settings:

• Preparation of lysis buffer
• 2 volumes 98 % glycerol (v/v)
• 1 volume 0.44 M NaOH
• 1 volume 5 % SDS (w/v)
• Protocol
• Resuspend cells in 20 µL H₂O
• Add 5 µL Loading buffer
• Seperate in agarose gelelectrophoresis with supercoiled marker
• Elute from gel

• 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. You can add less glacial acetic acid and check the pH by pipetting some µL of the solution on pH paper. We observed slighty better performance for our reactions at pH = 7.9. 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

• 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
• Aliquot and flash-freeze. During summer we prepared this premix more than one time, and we observed that the age of the cofactor premix does not affect the cell free reaction as long as aliquots are stored at -80 °C.

• 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 per 1 g of wet cells 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 sonication directly.

The following strategy can be pursued if there is no access to a sonifier which can display its energy output in Joule per second or alike.

First, prepare multiple reference samples which are absolutely identical (see below for further requirements). Right before sonication, ensure the temperature of the liquid. Sonicate for a defined time interval. When sonication has ended, measure the temperature of the liquid again without any delay.

Perform at least triplicates for every interval. To calculate the power P of your sonifier in Joule per seconds and thereby calculate the necessary sonication time for a given setting, use the nearby equation. You need to consider the following:

• The Amplitude of the sonifier must be kept constant during experiment to eliminate the influence of viscosity or heating on the result
• The starting temperature must remain the same in all reference measurements
• The liquid as well as the vessel used in the reference experiment must be similar to the ones in the intended experiments

For our setting (see below), a sonication time of 290 s was appropriate for a total energy input of 800-900 J.

Our lysis procedure is described in the following section

Sonication of the pellet from Cell harvest with the HF-Generator GM 2070 in combination with UW 2070, SH 70G and MS73; setted at 70% amplitude and no cycles

• clean the sonifier tip with 70% (v/v) EtOH prior to usage
• resuspend the pellet in 1 mL S30 buffer per gramm pellet
• aliquot the cell suspension à 1500 µL in 2 mL reaction tubes
• for each sonication procedure, place one tube in an ice bath and place the sonifier tip in the suspension, so that it is briefly at 1/3 height of the tube
1. sonicate for 10 s
2. wait 10 seconds
3. repeat the previous two steps 28 times
• Centrifuge for 10 min at 12000x g and 4 °C.
• Carefully transfer the supernatant (cell extract) into a new tube. Take care to not disturb the pellet.
• Consider to conduct a run-off reaction at 37 °C, slightly shaking at 250-300 rpm, in order to digest endogenous DNA and mRNA. When using the extract for the first time, perform various run-off reactions, e.g. 20 min to 80 min in 10 min intervals. For us, 30 min gave the best results.
• Centrifuge a second time at 10000x g, 4 °C, 10 min after performing the run-off reaction.
• Transfer the supernatant (cell extract) into new tubes (we used 50-100 µL aliquots). Flash-freeze in liquid nitrogen and store at -80 °C.

An extensive analysis on sonication and run-off reaction parameters can be found in Kwon and Jewett 2015.

• To prevent RNase contamination, work with gloves all the time. It is strongly recommended to use filter tips or low-stick tips, respectively, and recently calibrated pipettes in order to work precisely with very low volumes. Do triplicates or quadrupates for a reliable data evaluation.

Materials needed

• reagents
• cell extract
• cofactor premix
• amino acids
• NTPs
• energy source and corresponding cofactors
• DNA template
• Mg- and K-glutamate solutions
• RNase free water
• any other additives
• other materials
• Depending on what protein is to be produced, a device capable of measuring fluorescence and/or luminescence. In our case this was a TECAN infinite M200 plate reader.
• Depending on what device you use (for example plate reader), a 386 black well plate and transparent sealing foil
• RNase free microcentrifuge tubes
• 70% EtOH for RNase decontamination

Procedure

1. Prepare a sheet in silico that contains all information you need for pipetting. For our purpose we created a suitable excel spreadsheet based on the one from Sun et al., 2013
2. Clean your working place thorough with 70% EtOH. Prepare RNase free micocentrifuge tubes. Let all aliquots thaw on ice.
3. Start to prepare your DNA samples by adding water, DNA, glutamate salts and any other item of choice to microcentrifuge tubes at room temperature.
4. Combine aliquots from 2. to a reaction buffer in a microentrifuge tube. Shortly vortex after the addition of each reagent and keep on ice.
5. At the latest after your reaction buffer is ready, prepare your measurement device (software program, temperature and so on).
6. Prepare a master mix made of reaction buffer and E. coli cell extract according to your spreadsheet, shortly vortex and keep on ice for now. From now on, do not waste excessive time with dreaming but work eagerly so that, as long as your template is missing, no energy in the buffer is wasted by cell extract proteins!
7. Add the calculated master mix amount to each of your DNA sample after carefully mixing the master mix. Attention: The master mix is viscous, avoid bubbles by pipetting the last drop to the tube wall.
8. Shortly vortex each sample and centrifuge for 30 s at 10000 g at room temperature. Thereby any residual sample will be brought down, and bubbles can be reduced.
9. Run the reaction
• in the microcentrifuge tubes or
• in a multi-well plate by transferring the samples into wells, sealing with appropriate foil to prevent evaporation. Optional: If you have a plate centrifuge, you can minimize bubbles in the plate with a short centrifugation step prior to measurement (4000 g, 30 s according to Sun et al., 2013)

• Attention: Consider a volume loss during pipetting. We routinely performed reactions at a final scale of 15 µL, but in our spreadsheet we calculated with 15.75 µL.
• Run the reaction at the desired temperature for a desired time. In most of our experiments we measured for 3 h and saw no further raise in fluorescence signl. In most cases this is 37 °C but optimal temperature varies depending on protein product. Some proteins are well produced over a wide range of temperatures, which is the case for sfGFP.
• Other considerations
• For best performance, a DNA plasmid of high purity and water of high quality are recommended. For sfGFP expression plasmids we found that plasmid mini-preparation kits, especially from Promega and Qiagen, provide sufficient DNA quality.
• Shaking can be helpful, especially when oxygen is needed for the reaction (see Caschera and Noireaux 2015). In our Tecan measurements we shaked 5 s with 1 mm amplitude every time before fluorescence was measured.

• 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.

• Required solutions:
• 5x EMSA buffer (100 mM Na₂HPO₄, 375 mM KCl, 25 % Glycerol, adjust ph to 8.0)
• EMSA running buffer (20 mM Na₂HPO₄, adjust ph to 8.0)
• 3 % agarose gel prepared with EMSA running buffer
• MgCl₂: 25 mM
• EDTA: 2,5 mM
• BSA: 10 mg/ml
• Dithiotreitol
• salmon sperm
• Cy3 labeled DNA
• protein
• Reaction:
• 0.05 pmol Cy3 labeled DNA
• 5 pmol purified protein of interest
• 4 µl 5x EMSA buffer
• 2 µl MgCl₂ final concentration: 2.5 mM
• 0.8 µl EDTA final concentration: 0.1 mM
• 1 µl BSA final amount: 10 µg
• 1 µl salmon sperm final amount: 1 µg
• optional: 5 mM DTT
• mix all components of the reaction carefully and add up with water to 20 µl H₂O.
• Incubate at room temperature in the dark for 15 min.
• Keep in mind that Cy3 is light sensitive, always keep your samples in the dark after addition of the DNA fragment.
• Apply reaction to 3 % agarose gel.
• Run gel at 80 V for approximately 80 min at 4 °C. (Adjust voltage and duration to size of your DNA fragement.)
• Detect Cy3 with a fluorescence scanner. In our case it was the Typhoon 8600 variable Mode Imager.

• 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.)

• PicoGreen Assay
• 99 µl TE-buffer (10 mM Tris-HCl, 1mM EDTA, pH 7.5) are mixed with 1 µL sample solution in a well of a black 96 well plate
• For the determination of the concentration, a standard row of the concentrations 1, 0.5, 0.25, 0.125 and 0.0625 and 0 µg/mL of lambda DNA is done.
• PicoGreen solutions are diluted in a ratio 1/200 in TE-buffer.
• 100 µl diluted PicoGreen solution is mixed with the diluted sample.
• The samples are measured in a Tecan Reader. They are excited at 480 nm and the fluorescence emission intensity is measured at 520 nm.

• You can detect immobilized DNA and 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.

• streak out 1 LB plate per device and control (for each biological replicate 1 plate)
• incubate LB plates over night at 37°C (19 hours)
• inoculate liquid cultures (100 mL flasks with 10 mL LB-medium)
• incubate at 37 °C, 300 rpm, 17 hours

• measure the OD600 of the overnight cultures
• dilute all samples to an OD600 of 0.5
• re-measure the OD600, if it is within 5 % of 0.5, proceed
• measure the samples with a plate reader (150 µl/ well, 96-well-plate, Excitation Wavelength : 475 nm, Emission Wavelength: 509 nm)