Add pABA (1 equiv) to MeCN (2 mL, 0.5 M)

Add 1 equiv trimethyl borate to mixture. Stir at 80C (100C?) for 5-24 hours.

Dilute reaction mixture with CH2Cl2 or EtOAc (organic solvent) and H2O (polar solvent).

Add MgSO4 (anhydrous) to remove H2O.

Heat mixed tubes at 93°C for 1 min in thermal cycler

Decrease heat of thermal cycler by 5°C every 2 min with small ∆T/sec in between steps

Once at room temperature, hold samples at 4°C for 15 min, then store samples at -30°C

Let all reagents/samples/standards equilibrate to RT.

Prepare enough fresh working reagent (WR) for all standards and samples to be measured using a 50:1 ratio of the kit reagents A:B.

Add reagent volume to each PCR tube.

Add 10 uL of standards or samples to the appropriate tube. Mix well by gentle vortexing.

Incubate standard and sample tubes at:

Prepare WN + WS buffers by adding appropriate amount of 100% ethanol as shown on bottle.

Carefully cut out piece of gel containing fragment of interest under longwave UV.

Add 300ul GEX for every 100mg gel collected to centrifuge tube and gel piece.

Incubate at 55˚C for 5-10min until gel has dissolved. Can smash with a pipette tip.

Place extraction column into collection tube and load 0.7ul mixture into column. Centrifuge at 5000rpm for 30s. Discard flow-through. Repeat until all mixture is used.

Wash column with 0.5ml (500ul) of WN buffer by centrifuging at 5000rpm for 30s. Discard flow-through.

Wash column with 0.5ml WS buffer by centrifuging for 60s at 5000rpm. Discard flow-through.

Centrifuge column at 12000rpm for 3min to remove residual ethanol.

Place column in new (storage) centrifuge tube. Add 15-30ul elution buffer onto center of membrane. Allow to stand for 2min.

Centrifuge for 50s at 12000rpm to elute DNA. Store at -20˚C. Find concentration collected with Nanodrop.

Spin down DNA tubes from Competent Cell Test Kit at 8,000-10,000rpm for 20-30 seconds

Thaw competent cells on ice for 10 minutes. Label one 2.0ml microcentrifuge tube for each concentration and pre-chill by placing empty tubes on ice.

Pipet 1uL of DNA into each microcentrifuge tube. Use a separate tube for each concentration.

Pipet 50uL of competent cells into each tube, flicking tube gently to mix. Incubate on ice for 30 minutes. Preheat waterbath to 42˚C.

Heat-shock cells by placing into waterbath for 1 minute.

Immediately transfer tubes back to ice and incubate on ice for 5 minutes.

Add 200uL of SOC per tube and incubate at 37˚C for 2 hours. Prepare and label agar plates.

Pipet 20uL from each tube onto appropriate plate and evenly spread mixture across the plate. Do triplicates of each tube if possible to calculate an average colony yield.

Incubate at 37˚C overnight or for 16 hours.

Count number of colonies against a dark background, using an average cell colony count if done in triplicates.

Competent cells have an efficiency of 1.5x10^8 to 6x10^8 cfu/ug DNA (cfu = colony-forming unit)

Use a pipette tip to scrape off a little bit of the semi-thawed glycerol. Put tip with cells into media

Put your glycerol stock back into the box in the -80°C freezer. Do not let it thaw completely! Thawing and freezing your stock repeatedly will kill your cells.

Put inoculated culture into shaking incubator to let culture grow.

1000 pmol oligo mixture for 200uL magnetic beads (500pmol/mg)

Resuspend in B&W to a concentration of 2mg per mL (2×10⁹ beads/mL)

Add equal volume of oligo (1000pmol) and incubate at room temperature for 1 h with gentle rotation

Resuspend in selection buffer 1×10⁹ beads/mL (1×10⁸ beads/mL for each subsequent round), so 2.0 mg in 2mL

Store at 4C. When use, resuspend completely (i.e. vortex etc.)

Dilute Target to 1mM in selection buffer

Sterile filter (used pore size of 0.22um), aquilot and store at -18 °C

DNA library (2-3nmol 1st round, all in following rounds) heated to 90 °C for 8min

At same time, wash streptavidin bead-capture oligo complexes (1x10^9 and 1x10^8 in following) around 3X w/ 500uL SB

Bead-capture oligo complexes resuspended in 300uL of the pretreated DNA library

Wash beads around 9X w/ 500uL SB to remove unbound DNA

Incubate DNA-bead complexes in 500uL SB at 28 °C for 15min w/ mild shaking

Wash around 7X w/ 500uL SB (to remove all remaining unhybridized or weakened DNA structures)

Suspend DNA-bead complexes in 300uL SB

Wash around 7 X with 500uL SB

Suspend DNA-bead complexes in 300uL target mixture

Use a magnet to separate out the beads, saving the remaining liquid

Save 10% of the flow-through as stock solution (labeling as necessary), use rest for PCR amplification in the following step.

Nanodrop, determine appropriate number of parallel PCR reactions (e.g. 15, 100uL each)

For Q5 Master Mix, set thermocycler to

<Gel electrophoresis on 2.5% agarose gel to confirm proper length of DNA product (if feel compelled to do so)>

Pool PCR product and precipitate with ethanol in presence of linear polyacrylamide [19]

Resuspend product in 100uL TE buffer

Run denaturing PAGE in TBE buffer

Elute DNA from gel with 2mM EDTA, 300 mM sodium acetate, and pH 7.8 (5.2)(around 5mL of this solution) -- adding acetic acid

Precipitate DNA with ethanol in presence of linear polyacrylamide [19]

Resuspend DNA in SB.

<goal of around 13 rounds>

cloning+transformation into chemically competent cells

+sequencing -- talk to Mark

Discuss SPR with Michaela

Archive roughly 10% of each round and save as stock solution

With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.

Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.

Transform 1ul of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.

Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.

Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.

Rinse 500ml cylinder with water then ethanol.

Measure 120ml ethanol, add to wN buffer.

Add 180ml ethanol to WS buffer.

Centrifuge liquid culture tubes at 4500rpm (max) for 2min. Be sure to balance tubes.

Resuspend cell pellet in 200ul MX1 buffer kept in fridge.

Add 250ul MX2 buffer and gently mix. Incubate at room temp for 1-5 min, don't vortex.

Add 350ul MX3 and gently mix immediately.

Centrifuge for 5-10 min at 14,500rpm.

Transfer supernatant carefully to column.

Centrifuge 30-60s at 5000rpm. Discard flow-through.

Wash column once with 500ul WN buffer by centrifuging for 30s at 9000rpm. Discard flow-through.

Wash column once with 700ul WS buffer by centrifuging for 30s at 9000rpm. Discard flow-through.

Centrifuge column at 13000rpm for 2min to remove ethanol residue.

Place column into new 1.5ml centrifuge tube, add 50ul Elution Buffer onto center of membrane.

Stand for 2min at room temp, then centrifuge for 30s at 13000rpm to elute DNA.

Store plasmid DNA at 4˚C or -20˚C.

Load 1ul molecular ladder into first row.

Mix 1.5ul purple loading dye, 1ul DNA, 7.5ul DI water on a sheet of parafilm.

Load each row you're going to run.

Plug in cables and switch on machine to +/- 100V for +/- 40min. Too high voltage will decrease resolution of results but will run more quickly.

Mix TAE and agarose (~1g powder per 200ml buffer (slightly less for long DNA)).

Microwave and mix until completely dissolved, pause microwaving when it bubbles.

Cool until can touch it. Add gel red and mix.

Carefully pour into box mold and cool at room temperature, covered with plastic wrap. Insert comb.

Before running gel, ensure TAE in box is just above gel (+/- 400ml).

Gibson Master Mix 2x: 5ul x 3 = 15ul

His tags: 1ul x 3 = 3ul

Digested vectors: 1ul

H2O: 3ul x 3 = 9ul

25g/L LB, 15g/L agar

Optional: 48˚C for 5min, 5 @ 45

50˚C for 15min, 10 @ 50

Optional: 52˚C for 5min, 5 @ 55

4˚C forever

Linearise vector plasmid where you want to insert new gene (eg using a restriction site).

Manually replace start codon if lost.

Select the gene you want to insert excl. stop codon and extract.

Select vector and extracted gene and click tools --> cloning --> Gibson assembly.

Ensure backbone = vector, box contains gene fragment. Choose min overlap length (18-22), min overlap Tm (48), calculation of Tm is based on protocol.

Add the non-DNA components to a new PCR tube

Add an appropriate amount of DNA (75 ng per component is recommended)

Fill to 20 μL with DI H₂O

For 1-4 fragments, incubate at 37°C for 1 hr and 55°C for 5 min. For 5-10 fragments, alternate between 37°C and 16°C for one minute each for 30 cycles, then incubate for 5 min at 55°C. For more than 10 fragments, consider going that extra mile and buying the optimized master mix.

Proceed directly to transfomation.

Let all reagents in the kit to completely warm to RT before opening.

1X Rxn buffer: Add 4 mL of 5x Reaction buffer (Component C, white cap) to 16 mL of dH2O.

10 U/mL Horseradish peroxidase stock soln: Dissolve HRP (Component D, yellow cap) in 1.0 mL of 1x Rxn buffer.

20 mM H2O2 working soln: Dilute ~3% H2O2 (Component E, red cap) into appropriate vol of 1X Rxn buffer to make 20 mM H2O2. Actual concentration of H2O2 is indicated on the label.

Prepare H2O2 standard curve. Dilute H2O2 containing samples in 1X Rxn buffer to produce H2O2 concentrations of 0-10 uM.

Prep working soln of 100 uM Amplex Red Reagent/HRP. Mix 50 uL of 10 mM Amplex Red reagent stock soln + 100 uL of 10 U/mL HRP stock soln, 4.85 mL of 1X Rxn buffer.

Begin rxn. Add50 uL of Amplex Red reagent/HRP working soln to each microplate well containing standards/controls/samples.

Incubate rxns at room temp for 30 mins, protected from light.

Use plate reader to detect fluorescence (excitation 530-560 nm, emission 590nm, absorbance 560nm).

For each point subtract value derived from no-H2O2 control

Estimate amount of inclusion body prep by subtracting weight of centifuge tube from total weight. Use 8 mL of Inclusion Body Solubilization Reagent per 1 g of wet inclusion body pellet.

Suspend pellet in appropriate amount of Inclusion Body Solubilization by vortexing or pipetting. Shake suspension of 30 min.

Remove cell debris by centrifugation at 27k x g (15k rpm) for 15 min.

Collect supernatant which contains the solubilized protein. For protein assay, use Coomassie Plus (Bradford) Protein Assay Kit. For SDS-PAGE analysis, remove denaturant by dialysis.

Collect bacterial cells that express protein of interest by centrifuging at 5k x g for 10 min.

Carefully remove the spent medium from the cell pellet. The cell pellet may be frozen or used fresh. Frozen cell pellet will give slightly higher yield of protein

Add CelLytic B at ratio of 10-20 mL per gram of cell paste. Mix well to completely resuspend the cells.

Incubate extraction suspension with vortexing/shaking at room temperature for 10-15 minutes to fully extract soluble proteins from cells

After cell extraction, centrifuge the extract at 16k x g for 10 mins to pellet insoluble material.

Carefully remove supernatant containing soluble protein fraction. Another round of extraction will yield more soluble protein if required - but will result in more dilute soluble protein sample

Analyze supernatant and insoluble fraction by SDS-PAGE and/or Western blot to determine which fraction contains protein of interest. For SDS-PAGE use 5-15 uL of each sample for gel.

Thaw ligase buffer in hand and vortex, then put immediately on ice.

Mix materials in PCR tubes with a total volume of 52.5ul

Set at 95˚C and cool at 0.1˚C on Jessica's thermocycler for 19 minutes

Dilute 1:10 if doing Gibson

Do Gibson protocol

Add 5ml LB broth to 15ml tube.

Add 5ul antibiotic.

Add +/- 1ul/scrape of cells.

Shake up and incubate at 37˚C at 180rpm.

Add agar powder (15g/L) to LB broth.

Autoclave.

Cool to 55˚C then add antibiotic. Pour into petri dishes.

Allow to harden and store inverted at 4˚C in the dark (antibiotic is light sensitive).

If agar solidifies too early, possible to microwave it but it may explode.

Read blank of 1ul elution buffer.

Run each sample, name at Sample ID, hit measure.

Want 260/280 + 260/280 ~ 2.

Record concentrations on stored tubes.

In a PCR tube, mix:

Maintain all on ice throughout process.

Gently mix tube.

Place in thermocycler and cycle at:

Final hold 4-10˚C.

Place all materials on ice to thaw. If needed, thaw Ligase 10x Buffer in hands and vortex until all white precipitate has disolved. Place back on ice for 2 min before adding to rxn mixture.

Add materials listed above with desired 80 µM DNA building block mix to PCR tube

Incubate in thermal cycler at 37°C for 1 hr

Add 1 µl T4 DNA Ligase to each tube -- vortex and spin down to mix if

Incubate at 16°C overnight

Carefully remove gel from box and place on scanner.

Select area on scanner.

Run scanner.

Use 1.5 mL of bacterial culture with OD600 of 0.5-1.0 and centrifuge the cells at full speed/7k for 2 min.

Remove spun medium and resuspend the cell pellet in 0.4 CelLytic B.

Carefully remove the soluble protein fraction from the cell debris. Additional extractions may be performed if required, but will make your soluble protein sample more dilute.

Analyze supernatant and insoluble fraction by SDS-PAGE and/or Western blot to determine which fraction contains the protein of interest.Fir SDS-PAGE, use 5-15 uL of each sample for gel.

Thaw cells on ice for 10min.

Add 1-5ul contianing 1pg-100ng plasmid DNA (eg from Gibson assembly).

Place mixture on ice for 30min. Do not mix.

Heat shock at 42˚C for 10s exactly.

Place on ice for 5min.

Pipette 950ul room temp SOC.

Place at 37˚C for 60min, shaking at 250rpm.

Warm selection plates to 37˚C.

Mix cells and perform dilutions.

Spread 50-100ul of cell dilution onto plates. Incubate overnight at 37˚C.

Thaw NEB 5-alpha competent E.coli on ice for 10 minutes (Half a stock is fine, half each subsequent amount)

Add 1-5ul of 1pg-100ng plasmid DNA to cell mixture. Flick tube to mix, do not vortex.

Place on ice for 30min. Do not mix.

Heat shock at 42˚C for 30s.

Place on ice for 5min.

Pipette 950ul room temp SOC into mixture (475 if using half stock).

Place at 37˚C for 60 minutes, shaking at 250rpm.

Perform several 10-fold dilutions (1:10, 1:100) of cells.

Plate 50-100ul of each dilution onto an antibiotic selection plate.

Incubate overnight at 37˚C.

Place electroporation cuvettes, microcentrifuge, and cuvette holder on ice.

Thaw NEB 5-alpha electrocompetent E.coli on ice for 10min.

Transfer 25ul of cells and 1ul DNA into microcentrifuge tube.

Transfer mixture to cuvette. Make sure all parts are cold to avoid cells exploding.

Electroporate with the following conditions:

Add 975ul SOC in cell box to cuvette, mixing gently, then place in round bottom culture tube.

Incubate at 37˚C, shaking at 250rpm for 1hr.

Dilute cells (1:10, 1:100) and spread 100-200ul onto antibiotic selection plate.

Incubate cells overnight at 37˚C.