Conjugate folic acid with ssDNA

Protocol

1. Dissolve 1.0 mmol folic acid, 1.2 mmol DCC, and 2 mmol NHS together in 20 mL DMF, avoiding light.
2. Keep reaction at room temperature for 1 day, avoid light.
3. Filter the reaction product. The filtrate was washed with 100 mL mixture of acetone and ether (3: 7). The precipitate (folic acid NHS ester) will be a yellow solid product.
4. Dissolve 2.5 mmol MES in 30 mL ddH2O and adjust pH with 2N NaOH to 5.54. Add ddH2O until the volume is 50 mL, completing the preparation of MES buffer (50 mM, pH 5.54).
5. Completely dissolve 5.4 mg folic acid NHS ester in 1 mL DMSO, and then add MES buffer to 10 mL, giving the final concentration of 1 mM.
6. Take 10 μL of 100 μM ssDNA-NH2 and 10 μL folic acid NHS ester solution and add 80 μL MES buffer to form a 100 μL FA-ssDNA solution.
7. Incubate reaction at room temperature for at least 4 hours.
8. Dialysis the 100 μL FA-ssDNA solution through MWCO 3.5KDa dialysis membrane with 0.1X MES buffer with 1000X volume. Incubate at room temperature for 24 hours.
9. The solution in the membrane is the final product FA-ssDNA.

Conjugating NHS-ester modifications to poly-(methyl methacrylate)

Protocol

1. Wash the PMMA with ddH2O and dry the surface, clean it with Oxygen Plasma Cleaner HARRICK PLASMA (30W, 20 min).
2. Dissolve 2.5 mmol MES in 30 mL ddH2O and adjust pH with 2N NaOH to 5.54. Add ddH2O until the volume is 50 mL, completing the preparation of MES buffer (50 mM, pH 5.54).
3. Dissolve 2.1 mmol EDC and 0.87 mmol NHS in 10 mL MES buffer, then completely soak the PMMA that has been cleaned by oxygen plasma into the solution for 30 minutes.
4. Take out the PMMA and dry it, and store it in a dry cabinet to avoid contact with moisture.

Gibson assembly

Protocol for 2-3 Fragment Assembly

1. Prepare 10μl mixture of linear vector: insert = 1:3 (pmole) on ice.
2. Add 10 μl Gibson Assembly Master Mix (2X) to 10μl mixture on ice.
3. Incubate the reaction at 50°C for 40 mins.
4. Transform the E. coli with the Gibson assembly mixture.

Prepare LB agar plate

Protocol

1. Mix 25g/L LB Broth and 15g/L agar in a serum bottle.
2. Autoclave at 121°C, 2 atm for at least 20-minute liquid cycle.
3. Heat the molten gel mix by partially submerging the bottle in the 60 ℃ water bath.
4. Add antibiotics with proper concentration, and mix by swirling.
5. Pour into plates (~15ml/plate), covering the surface and avoiding bubbles.
6. Let plates cool with the lids ajar.

Transformation

Materials

• Competent cells ECOS™ FYE 307/FYE678/FYE207/FYE508 (YB Biotech).

Protocol

1. For the ligation product, add competent cells with 3 times of ligation reaction volume, and incubate on ice for 5 mins. For intact plasmid, mix 50 ng plasmid with 10 μl competent cell.
2. Heatshock the competent cells at 42°C for 45 seconds.
3. Plate to LB-agar plate.

Colony PCR

Protocol
-Preparation

• For each colony, prepare one tube for PCR and one tube for saving bacterial stock.
• Add 5 μl ddH2O into each 200 μl PCR tube.
• Add 50 μl TB with antibiotics into each 1.5 ml tube to save the bacterial stock.
1. Pick colonies for PCR and stock
• Using a tip, pick a single colony, and gently mix it in 5 μl of ddH2O in the PCR tube, immediately followed by mixing it in the TB with antibiotics in the 1.5 ml tube.
2. Prepare PCR mix (total volume:15μl )
• For each colony, prepare PCR mix with the following table.

component	volume
GoTaq® Green Master Mix	10 μl
Primer (Forward)	0.5 μl
Primer (Reverse)	0.5 μl
ddH2O	4 μl
Total	15 μl

• Add 15μl mixture to each PCR tube.
• Run PCR

Bacteria stock

Protocol

1. Mix the 50% glycerol and bacterial culture with 1:1 volume.
2. Store the mixture in the -80°C freezer as bacterial stock.

Agarose Gel extraction （Geneaid No.DFH300）

Protocol

Step 1: Gel Dissociation

• Transfer 300 mg (about 3 wells) of gel slice to 1.5 ml microcentrifuge tube.
• Add 500 μl of Gel/PCR Buffer, vortex, and incubate at 55-60°C for 10-15 minutes.
• Add 10 μl of 3M Sodium Acetate if turned to purple.

Step 2: DNA Binding

• Place DFH column in 2 ml collection tube.
• Transfer 800 μl sample mixture to the DFH column.
• Centrifuge (14-16000) for 30 sec.
• Discard the flow-through and place the DFH column back in the 2 ml collection tube.
• If the sample mixture is more than 800 ul, repeat the DNA Binding step.

Step 3: Wash

• Add 400 μl W1 Buffer into DFH column.
• Centrifuge (14-16,000 x g) 30 sec then discard flow-through.
• Add 600 μl Wash Buffer (make sure alcohol is added) into the DFH column, stand at room temperature for 1 min, centrifuge (14-16000 x g) for 30 sec then discard flow-through.
• Place the DFH column back and centrifuge (14-16000 x g) for 3 min to dry the column matrix.

Step 4: DNA Elution

• Transfer the dried DFH column to a new 1.5 ml microcentrifuge tube.
• Add 20 μl ddH2O into the center of the column matrix.
• Let stand for at least 2 minutes to ensure the water is completely absorbed.
• Centrifuge (14-16,000 x g) for 2 minutes to elute the purified DNA

Plasmid extraction (Geneaid No.PDH300)

Protocol

1. Draw 1.5 ml from a 2 ml bacterial culture tube into a new 1.5 ml tube, leaving the remaining 0.5 ml in the test tube.
2. Centrifuge the new 1.5 ml tube for 1 minute at 14000 rpm. Use a pipette to aspirate the supernatant after centrifugation (be careful not to aspirate the plasmid layer at the bottom).
3. Tilt the rack and use the tube to scrape it to disperse the bacteria in the tube.
4. Add 200 μl of PD1 and vortex once to mix the liquid.
5. Add 200 μl of PD2. Do not vortex to avoid DNA breakage. Instead, use two racks to sandwich the tube and shake it up and down 10 times.
6. Insert the pipette tip beneath the liquid surface and add 200 μl of PD3.
7. Centrifuge the tube for 3 minutes at 5000 rcf (relative centrifugal force) and place the PDH Column into a 2 ml Collection Tube. If proteins still remain suspended in the upper-middle layer, centrifuge again for 3 minutes at 15000 rpm.
8. Suck up 600 μl of the liquid containing DNA into the PDH column. Discard the 1.5 ml tube. (Do not aspirate the white proteins; if suspended proteins are aspirated, first draw 400 μl, then centrifuge the tube for 3 minutes at 15000 rpm, and then aspirate the remaining 200 μl).
9. Centrifuge the PDH column for 30 seconds at 15000 rpm and discard the flow-through.
10. Add 400 μl of W1 to the PDH column, centrifuge for 30 seconds at 15000 rpm, and discard the flow-through.
11. Add 600 μl of Wash Buffer to the PDH column, and centrifuge for 30 seconds at 15000 rpm (in the process, get a 1.5 ml tube ready). After centrifugation, discard the flow-through.
12. Centrifuge the dry PDH column with no added liquid for 3 minutes at 15000 rpm. (ensure the PDH column is dry).
13. Place the PDH column into a 1.5 ml tube, add 20 μl of Elution Buffer (can be replaced with 60°C ddH2O), and time for 2 minutes to ensure DNA binds to the Elution Buffer. (Elution Buffer can be replaced with 60°C ddH2O; it's recommended to heat ddH2O to 60°C for better DNA binding).
14. Centrifuge the 1.5 ml tube containing the PDH column for 2 minutes at 13000 rpm.

Rolling Circle Amplification (phi29-XT RCA Kit) (NEB #E1603)

Protocol

1. Prepare reactions without phi-29-XT DNA polymerase as described in the table below. Mix thoroughly, but gently, by pipetting or vortexing. Centrifuge briefly to collect solutions to the bottom of the tube.
2. Incubate in a thermocycler, with the lid set at 100°C, for 3 minutes at 95°C. Then allow samples to cool to room temperature.
3. Place samples on ice and add 2 µl of phi29-XT DNA Polymerase to each sample. Mix thoroughly, but gently, by pipetting or vortexing. Centrifuge briefly to collect solutions to the bottom of the tubes.
4. Incubate in a thermocycler with the lid set at ≥ 75°C, for 2 hours at 42°C, followed by 10 minutes at 65°C to inactivate the DNA polymerase. The RCA products can be kept at 4°C overnight or at -20°C for long-term storage.

Bacterial protein induction

1. Preparation
• Cultured 3 ml bacteria in TB-antibiotics at 37℃ overnight.


2. Refresh
• Quantify and dilute the grown bacteria to OD 600 value around 0.02.
• Refresh the diluted bacteria at 37℃ for 3 hrs.
• Quantify the OD600 value of refreshed bacteria. If OD 600 value reaches 0.2~0.4, store 1 ml of refreshed bacteria as the non-induction sample, If not, then put the refreshed bacteria back to 37℃ for one more hour.


3. Induction
• Induced the protein expression in the rest of the refreshed bacteria by 1mM IPTG at 37℃ for 6 hrs.
• Quantify the OD600 value of induced bacteria. If OD600 reaches 0.2~0.4, save all bacteria as the induction sample.


4. Storage
• Non-induction and induction samples are centrifuged at 5000 rpm for 3 mins.
• Remove the supernatant.
• Directly store the pellet in -20℃, or lyse the pellet with 100μl 1x sample buffer and store at -20℃.

Bacterial protein extraction

• Solution preparation

Protocol

1. Resuspend 1.5 mL pellet of bacteria cell culture in 0.5 ml lysis buffer.
2. Incubate 30 minutes on the ice.
3. Sonication 3x10" till the sample is no longer viscous.
4. Centrifuge 14000 rpm for 10 min at 4°C.
5. Collect the supernatant to a new tube.
6. Store sups and the dried pellet at -20°C.

SDS-PAGE

Materials & protocol

• Gel preparation

Lower Gel-For 2 Gel
Percentage	15%	12%	10%	7.5%
ddH2O	2.35ml	3.35ml	4.0ml	4.85ml
Tris buffer (1.5m, pH 8.8)+10% SDS	2.5ml	2.5ml	2.5ml	2.5ml
30% (29:1) Acrylamide: Bisacrylamide	5.0ml	4.0ml	3.33ml	2.5ml
10% APS	100μl	100μl	100μl	100μl
TEMED	10μl	10μl	10μl	10μl
Vtotal	10μl

Upper Gel-For 2 Gel
Percentage	5%	4%
ddH2O	1.75ml	1.85ml
Tris buffer (1.5m, pH 8.8)+10% SDS	0.75ml	0.75ml
30% (29:1) Acrylamide: Bisacrylamide	0.5ml	0.4ml
10% APS	30μl	30μl
TEMED	5μl	5μl
Vtotal	3μl

• Solution Preparation

Fixing solution
Ethanol	150 ml
Glacial acetic acid	50ml
ddH2O	300ml
Total	500ml

Staining solution
Methanol	150 ml
Glacial acetic acid	50ml
Coomassie brilliant blue	1 g
ddH2O	300ml
Total	500ml

Distaining solution
Methanol	150 ml
Glacial acetic acid	50ml
ddH2O	300ml
Total	500ml

1. Gel preparation
• Wipe glass plates and spacers and assemble them in a gel casting apparatus.
• Mix components for the resolving gel and pour into the gel plate.
• Add 200 μl ddH2O on the top of the resolving gel to flatten the resolving gel.
• Until the resolving gel is solidified, clean up the ddH2O on the resolving gel.
• Mix components for the stacking gel, and pour into the gel plate.
• Insert the comb into the top of the spacers, and then wait for the gel to solidify.


2. Gel electrophoresis
• Add 1x running buffer to the running tank and put the cast gel into it.
• Load 10 μl marker into the first well, and load 20 μl protein samples per well.
• The gel will run at 80 V until the dye front is migrated into the running gel (about 20 minutes), and increase to 100 V until the dye front reaches the bottom of the gel (about 90 minutes).


3. Gel fixing, staining, and distaining
• Put gel into gel-fixing solution for 1 hour.
• Stain the gel with staining solution for 20 minutes.
• Distain the gel for about 1.5~2 hours until the bands are clear enough.

Protein purification (small-scale protein purification)

Marerial

• His60 Ni Superflow Resin (TaKaRa)

Protocol

• These concentrations of Imidazole are not fixed, based on your experiment requirements. The basic principle is from low concentration to high concentration.

Step 1: Replace of storage buffer of Ni-sepharose with PBS

• Take 100 μl slurry Ni-sepharose beads, spin down at 600 rpm for 3 minutes, and discard the storage buffer.
• Add 600 μl PBS (>with 10mM Imidazole, pH=7.4), and Vortex to ensure complete resuspension of the beads.
• Centrifuge for 600 rpm to precipitate the beads, and discard the supernatant.
• repeat 3 times, then discard the PBS supernatant.

Step 2: Binding

• Add 400 μl lysate (overload) to the resin, rotate for 30 minutes, and centrifuge at 1000 rpm for 3 minutes to precipitate the beads.
• Transfer the supernatant to a new 1.5 ml tube and save it as the flow-through sample.

Step 3: Wash

• Wash precipitated beads with 600 μl PBS (with 100 mM Imidazole, pH=7.4) by rotating for 10 minutes.
• Centrifuge at 1000 rpm for 3 minutes to precipitate the beads and save the supernatant as washed samples.
• The wash times depend on your experiment demand (suggest 2~3 times)

Step4: Elution

• Eluted the binding protein from precipitated beads by adding 600 μl PBS (with 600mM Imidazole, pH=7.4), and rotating for 10 minutes.
• Centrifuge at 1000 rpm for 3 minutes to precipitate the beads and save the supernatant for eluted samples.

Protein extraction from Inclusion Bodies

Material

• 50 ml Lysis buffer

component	volume
50 mM Tris (pH 8.0)	2.5 ml
10% Glycerol	10 ml
0.1% Triton	0.05 ml
ddH2O	37.45ml
Total	50ml

Protocol

1. Add 0.5 ml lysis buffer into 1.5 ml tube containing bacterial pellet.
2. Settle the 1.5 ml tube into ice for 30 minutes.
3. Use a sonicator to disrupt the cell (Please search for instrument usage instructions or contact experts in this field), making the protein inside release to the supernatant (sonication process should be operated on ice).
4. Centrifuge at 14,000 rpm for 10 minutes, 4°C. The sample will be separated into a supernatant layer and a pellet. Our target protein will be present in the pellet of the sample.
5. Wash the inclusion body pellets with 500 μl lysis buffer containing 1% Triton X-100, vortex to resuspend the pellets, and centrifuge at 1000 rpm for 3 minutes, the wash times depend on your experiment demand (suggest 2~3 times).
6. Solubilize the purified inclusion bodies into 8M urea with PBS buffer and 10 mM DTT for 30 minutes. Some inclusion bodies are very difficult to solubilize and you might want to leave them to dissolve overnight.
7. Run a gel to check for the success of the inclusion body extraction.

Cell culture

Protocol

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions
3. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium. and spin at approximately 125 xg for 5 to 7 minutes.
4. Resuspend the cell pellet with the recommended complete medium (see the specific batch information for the culture-recommended dilution ratio). and dispense into a 25 cm2 or a 75 cm2 culture flask. It is important to avoid excessive alkalinity of the medium during the recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
5. Incubate the culture at 37°C in a suitable incubator. A 5% CO2 in air atmosphere is recommended if using the medium described on this product sheet.

Cell passage

Materials

• 0.25% (w/v) Trypsin- 0.53 mM EDTA solution.
• growth medium

Protocol

1. Remove and discard existing culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to the flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 15 minutes).
4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.
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