EXPERIMENTS

gBlocks

1. Before opening the tube, spin it down in a microcentrifuge for 3–5 seconds to ensure the DNA is in the bottom of the tube.

2. Add IDTE to reach a final concentration of 50 ng/µL.

3. Vortex briefly.

4. Incubate at approximately 50°C for 15–20 minutes.

5. Ensure that the pellet of dried DNA is completely dissolved in IDTE

6. Briefly vortex and centrifuge

Resuspension of Primers

1. Find the oligo yield information (in nmol) on the tube label or specification sheet.

2. Multiply this number by 10.

3. The resulting product is the amount of buffer needed, in µL, to prepare a 100 µM solution.

4. Dilute the 100 µM stock to 10 µM as the working stock for all PCR reactions.

Q5® High-Fidelity PCR

1. Transfer contents into a 0.2 mL PCR tube according to the following recipe:

Component	25µL Reaction	50µL Reaction (for gel extration)
Q5 High-Fidelity 2× Master Mix	12.5 µL	25 µL
10 µM Forward Primer	1.25 µL	2.5 µL
10 µM Reverse Primer	1.25 µL	2.5 µL
Template DNA	20 ng	20 ng
Nuclease-Free Water	to 25 µL	to 50 µL

2. Set the PCR Thermocycling Conditions:

Step	Temperature	Time
Initial Denaturation	98°C	30 seconds
25-35 Cycles	98°C	10 seconds
	65°C	30 seconds
	72°C	30 seconds
Final Extension	72°C	2 minutes
Hold	4°C	-

Restriction Enzyme Digestion

1. Transfer contents into a 0.2mL PCR tube according to the following recipe:

Component	50µL Reaction
DNA	1µg
10X CutSmart Buffer	5µL (1X)
NheI-HF	1.0µL (20 units)
EcoRI-HF	1.0µL (20 units)
Nuclease-free Water	to 50µL

(2.) When further manipulation of DNA is required:

Heat inactivation (65°C for EcoRI-HF® & 80°C for NheI-HF®).
Remove enzyme by using Takara's Miniprep DNA Purification Kit # 9085.

Pre-heat the Elution Buffer or sterilized water to 60℃ can increase the elution efficiency.
For highest recovery, Incubate for 1 and a half hour / as long as possible at 37℃.

Remark: Incubate for 3 hours or overnight for better yield.

DNA Fragment purification

1. Add a 3X volume of Buffer DC to the PCR or other enzymatic reactions.

If the required volume of Buffer DC is less than 100 μL, add 100 μL of Buffer DC

Vortex briefly to mix the sample.

2. Place a Spin Column into a Collection Tube.

3. Pipette the sample from Step 1 into the Spin Column. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through.

Note: For improvement the recovery of DNA, transfer the flow-through to Spin Column and centrifuge again.

4. Add 700 μL Buffer WB to Spin Column. Centrifuge at 12,000 rpm for 30 seconds. Discard the flow-through.

Note: Make sure that the volume of 100% ethanol indicated on the bottle label has been added to the Buffer WB.

5. Repeat Step 4.

6. Place the Spin Column back into the Collection Tube. Centrifuge at 12,000 rpm for 1 minute.

7. Place the Spin Column into a new clean 1.5 ml tube. Add 25 −30 μL Elution Buffer or sterilized water to the centre of the membrane.

Let it stand for 1 minute at room temperature.

Note:

Pre-heat the Elution Buffer or sterilized water to 60℃ can increase the elution efficiency.
For highest recovery, Incubate for 1 and a half hour / as long as possible at 37℃.

Centrifuge at 12,000 rpm for 1 minute at room temperature to elute DNA.

Ligation

1. Transfer contents into a 1.5 mL microcentrifuge tube according to the following recipe on ice:

Component	20µL reaction
T4 DNA Ligase Buffer (10X)	2µL
Vector DNA	Varies
Insert DNA	Varies
T4 DNA Ligase	1µL
Nuclease-free water	to 20µL

2. Use NEBioCalculator to calculate molar ratios.

3. The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.

4. Gently mix the reaction by pipetting up and down and microfuge briefly.

5. Incubate at 16°C overnight or room temperature for 10 minutes.

Inoue transformation

1. Thaw the cells, then aliquot 100 μL of cells into a 1.5 ml microcentrifuge tube.

2. Add 10ng of DNA into corresponding tubes.

3. Incubates for 30 minutes on ice.

4. Heat shock at 42℃ for 45 seconds.

5. Incubates for 30 minutes on ice.

6. Add SOC medium so total volume is 1 mL.

7. Recover cells for 45 minutes in shaker. at 37°C at 270 r.m.p.

8. Centrifuge cells at 3000×xg for 3 minutes.

9. Remove all liquid medium from the tube.

10. Resuspend in 100 ul of LB medium.

11. Spread plates using glass plates.

Electrotransformation of E. coli

Preparation of electrocompetent cells

1. Dilute a fresh overnight culture from 1:1000 to 1:50 into 1 L of fresh SOC medium without minerals.

2. Grow cells at 18°C at 270 r.m.p.

3. Harvest the cells in the early exponential phase (OD600 = 0.4) by centrifugation (4°C, 3200×g, 10min).

4. Chill pulsing buffer on ice.

5. Remove the medium.

6. Wash the pellet twice in 1 L of cold pulsing buffer (PB) and centrifuge (4°C, 6000g, 10min).

7. Add 10mL of cold PB to the cells.

8. Cells can now be used.

Storage of electrocompetent cells at room temperature

1. Instead of adding PB to cells, add an equal amount of cell storage buffer.

2. Thoroughly mix together so that they form an emulsion-like solution.

3. Aliquot the cells into 1.5 mL tubes.

4. Place the tubes into a vacuum desiccator.

5. Apply vacuum using a vacuum pump at 37°C for at least 10 hours such that the water evaporates.

6. The resulting tubes can be stored at room temperature or lower.

7. Add cold PB to reactivate the cells.

Electropulsation

1. Mix 30-40 μL of cells with 1-5 μL of DNA in the electropulsation cuvette. Make sure there are no bubbles in the cuvette.

2. Apply pulse of 3-7 milliseconds at 6-9 kV/cm.

Expression of transformants

1. Immediately after the pulsation, add 1 mL of SOC medium. Shake the cuvette to mix.

2. Put the mixture on a sterile tube and incubate for 1h at 37°C. Shaking is optional.

3. Plate the cells on solid medium with ampicillin, usually adding 100μL of cells is sufficient.

Glass Bead Plating

1. Add 6 beads on either side of each plate.

2. Flicker tube until cells are uniformly distributed.

3. Gently pipette a dilution of outgrowth onto the centre of pre-warmed plate.

4. Place up to 4 plates on a folded paper towel square.

5. Vigorously move the plates forward and backwards. Make two complete 360° rotations, with stops every 90 degrees.

6. Allow plates to sit for 2 minutes.

7. Pour off the beads into a beaker.

8. Incubate plates at 37°C.

Plasmid Purification

1. Growth of bacterial cultures. Pick a single colony from a freshly streaked selective plate into 1 - 4 mL of liquid culture containing the appropriate selective antibiotic. Incubate at 37℃ overnight with vigorous shaking. (Growth for 12 - 16 hours and do not more than 16 hours, otherwise the bacterial cells will be hard to lyse and the yield of plasmid DNA will be reduced.)

Note: The culture volume should not be excessive because excessive bacteria will decrease lysis efficiency and result in poor purity of plasmid DNA.

2. Use 1 - 4 mL of the E. coli culture. Centrifuge at 12,000 rpm for 2 minutes to harvest the cell. Discard the supernatant.

3. Add 250 μL Solution l (containing RNase A). Resuspend the bacterial cell pellet completely by pipetting up and down.

Note: Be sure that the bacteria are completely resuspended, and no cell clumps remain before addition of Solution ll.

4. Add 250 μL Solution ll and mix gently by inverting the tube 5 - 6 times to completely lysis the cell until the solution becomes viscous and slightly clear.

Note: Do not allow the lysis reaction to proceed more than 5 minutes.

5. Add 350 μL of 4℃ precooling Solution lll and mix immediately and thoroughly by inverting the tube 5 - 6 times until a compact white pellet has been formed. Incubate at room temperature for 2 minutes.

6. Centrifuge at 13,200 rpm at room temperature for 10 minutes.

Note: Centrifuging at 4℃ is not recommended for precipitation.

7. Apply the supernatant from Step 6 into a new clean 1.5 mL tube by precise pipetting. Centrifuge at 13,200 rpm at room temperature for 10 minutes.

Note: The white precipitate will clog the spin column, reducing the yield.

8. Place a Spin Column in a Collection Tube.

9. Apply the supernatant from Step 6 onto the Spin Column by pipetting. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through with the aid of pipette.

10. Pipette 500 μL of Buffer WA onto the Spin Column. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through with the aid of pipette.

11. Pipette 700 μL of Buffer WB onto the Spin Column. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through with the aid of pipette.

Note: Make sure that the amount of 100% ethanol indicated on the bottle label has been added to Buffer WB.

12. Repeat Step 11.

13. Place the Spin Column back into new 1.5 mL tube without a lid. Centrifuge at 13,200 rpm for 5 minutes. Discard the flow-through with the aid of pipette.

14. Place the Spin Column back into 1.5 mL tube without a lid. Centrifuge at 13,200 rpm for 3 minutes. Discard the flow-through with the aid of pipette.

Note: Residual ethanol from Buffer WB may inhibit subsequent enzymatic reaction.

15. Place the Spin Column in a new clean 1.5 mL tube. Add 50 μL Elution Buffer or sterile purified water to the center of the Spin Column membrane. Incubate for 1 and a half hour / as long as possible at 37℃. Note: Pre-heat the Elution Buffer or sterile purified water to 60℃ will improve the elution efficiency.

16. Centrifuge at 13,200 rpm for 1 minute to elute DNA.

Colony PCR

1. Transfer contents into a 0.2 mL PCR tube according to the following recipe:

Component	25μL reaction	50μL reaction
10µM Forward Primer	0.5µL	1μL
10µM Reverse Primer	0.5µL	1μL
DNA	variable	variable
OneTaq 2X Master Mix with Standard Buffer	12.5μL	25μL
Nuclease-free water	to 25μL	to 50μL

2. Use a sterile toothpick to pick up individual colonies and dip into each reaction tube.

3. Create a stock of each individual colony by streaking the toothpick onto another agar plate containing the appropriate antibiotics and grow overnight.

4. Set the PCR Thermocycling Conditions:

Step	Temperature	Time
Initial Denaturation	94°C	2 minutes
30 Cycles	94°C	30 seconds
	54°C	60 seconds
	68°C	60 seconds
68°C	2 minutes
68°C	10°C	-

Gel Electrophoresis

1. Measure 0.2g of agarose.

2. Mix agarose powder with 20 mL 1xTAE in a microwavable flask.

3. Microwave until the agarose is completely dissolved.

4. Dilute the GelGreen® 10,000X stock reagent into the molten agarose gel solution at 1:10,000 and mix thoroughly. GelGreen® can be added while the solution is still hot.

5. Cast the gel and allow it to solidify. Load samples and run the gels (100V, 30 mins).

6. Image the stained gel with a 254 nm UV transilluminator or 490nm to 500nm visible light imaging system with a long path green filter.

Gel Extraction

The entire procedure should be executed with careful pipetting. Protocol in detail is as below.

1. Excise the agarose gel slice containing the DNA fragment with a clean, sharp scalpel under ultraviolet illumination. Cut as close to the DNA as possible to minimize the gel volume and increase the recovery rate of DNA.

Note: DNA should not be exposed to ultraviolet illumination for a long time in case of DNA damage.

2. Cut the gel into small pieces by cutting the gel. The gel melting time in Step 6 can be shortened and the recovery rate of DNA can be increased.

3. Add 600 μL Buffer GM to gel for melting.

4. Mix well and melt the gel at room temperature (15 - 25℃). Intermittent vortexing is essential to accelerate gel solubilization.

Note: Gel must be completely dissolved, or the DNA fragment recovery will be reduced. Extend the melting time when the gel concentration is high.

5. After the gel has been completely molten, check the color of the solution. If the color becomes orange or pink from yellow, add 10 μL of 3 M sodium acetate (pH5.2) to the solution and vortex well until the solution return to yellow. When the DNA fragment is smaller than 400 bp, add further with isopropanol to final concentration of 20%.

6. Set a Spin Column into Collection Tube.

7. Transfer the solubilized agarose from Step 7 into the column. Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through. Transfer the flow-through to spin column again and centrifuge again.

8. Add 700 μL of Buffer WB into the Spin Column. Centrifuge at 12,000 rpm for 30 seconds. Discard the flow-through with the aid of pipette.

Note: Make sure that the amount of 100% ethanol specified on the bottle label has been added to the Buffer WB.

9. Repeat Step 8.

10. Place the Spin Column back into new 1.5mL tube without a lid. Centrifuge at 13,200 rpm for 5 minutes. Discard the flow-through with the aid of pipette.

11. Place the Spin Column back into 1.5mL tube without a lid. Centrifuge at 13,200 rpm for 3 minutes. Discard the flow-through with the aid of pipette.

12. Place the Spin Column into a new 1.5 mL tube. Add 30 μL of Elution Buffer or sterile distilled water to the centre of the membrane. Let it stand for 1 and a half hour / as long as possible at 37℃.

Note: Pre-heat the Elution Buffer or deionized water to 60℃ can improve elution efficiency.

14. Centrifuge at 13,200 rpm for 1 minute to elute the DNA.

Expression Using T7 Express lysY/Iq

1. Transform expression plasmid into T7 Express lysY/Iq. Plate on antibiotic selection plates and incubate overnight at 37°C.

2. Resuspend a single colony in 250 mL conical flask with liquid culture with Ampicillin.

3. Incubate at 37°C until OD600 reaches 0.4 - 0.6.

4. Induce with 40 µl of a 100 mM stock of IPTG (final concentration of 0.4 mM) and induce at 15°C overnight.

Purification of target protein

Preparation of Regenerated Amorphous Cellulose (RAC)

1. Dissolve 2 g of microcrystalline cellulose in 6 mL distilled water to a 500-mL centrifuge tube.

2. Slowly add 100 mL of ice-cold 86 % H3PO4 to the slurry with vigorous stirring

3. Incubate the mixture 1 h on ice with occasional stirring.

4. Add 400 mL of ice-cold water at a rate of 5 mL per addition with vigorous stirring.

5. Centrifuge the precipitated cellulose at 3,000×g for 10 minutes at 4°C.

6. Discard supernatant.

7. Resuspend the pellet in ice-cold water and centrifuge at 3,000×g for 10 minutes at 4°C. Discard supernatant.

8. Repeat step 7 four times.

9. Resuspend the cellulose pellet with 5 mL of 2 M Na2CO3 and 450 mL of ice-cold distilled water.

10. Centrifuge the solution at 3,000×g for 10 minutes at 4 °C Resuspend the pellet in distilled water.

11. Repeat step 9 once or until the solution reaches pH 5–7.

12. Store the RAC slurry as a 10 g RAC/L suspension solution at 4 °C

Autolysis of E.coli

1. Harvest the cells by centrifugation at 3960×g for 15 minutes at 25°C.

2. Discard supernatant and remove all traces of growth medium.

3. Resuspend cells immediately in 62.5 mL deionised water.

4. Exactly 15 seconds later, add 62.5 mL 1 mM EDTA solution.

5. Centrifuge at 3960×g for 15 minutes to recover the cell lysis solution.

6. Discard the precipitate.

Column Purification

1. Pack a RAC column by adding RAC solution in a glass column.

2. Load the cell lysis solution into the column.

3. Wash the column with 5-fold RAC volume of 50mM Tris-HCL to wash impurities in the RAC matrix.

4. Elute the purified protein by adding 4-fold RAC pellet volume of 80 % glycerol. Collect the proteins in multiple fractions.

5. Store at -20°C.

Centrifuge Purification

1. Mix the cell lysis solution with the RAC slurry. Ratio of protein to slurry should be 3.0 μmol to 1 g.

2. Incubate the mixture at 4°C for 10-15 minutes.

3. Centrifuge the mixture.

4. Resuspend the pellet in a fivefold RAC volume of 50 mM Tris–HCl buffer.

5. Centrifuge again.

6. Resuspend the RAC pellet in a 4-fold RAC pellet volume of 100 % glycerol.

7. Elute the purified protein by centrifugation 12,000×g and recover the pure protein in the supernatant.

8. Store at -20°C.

SDS-PAGE

Sample Preparation

1. Determine the appropriate concentration of sample to load.

2. Dilute the sample with sample buffer with added reducing agent. 2x: dilute 1part sample with 1part sample buffer.

3. Heat the diluted sample at 90–95°C for 5 minutes.

Setting up the Cell and running gel

1. Remove the gels from the storage pouch and prepare them for assembly.

2. Remove the comb of the cell.

3. Remove the tape under the cell.

4. Rinse the well of the cell with running buffer.

5. Set the electrode assembly to the open position.

6. Place the gel cassettes into the electrode assembly. Two cassettes are required to create a functioning assembly; when using 1 or 3 gels, use the buffer dam (included with the cell)

7. Place the first cassette with the short plate facing inward and so the gel rests at a 30° angle from the centre of the electrode assembly. Make sure the electrode assembly remains balanced.

8. Repeat for the second cassette.

9. Gently push both gels toward each other, such that they are against the green gasket that is built into the electrode assembly.

10. Align the short plates to ensure the edge sits just below the notch at the top of the green gasket.

11. While gently squeezing the gel cassettes against the green gaskets (maintaining constant pressure and with both gels in place), slide the green arms of the clamping frame one at a time over the gels, locking them into place.

12. The wing clamps of the electrode assembly lift each gel cassette up against the notch in the green gasket, forming a seal. Check again that the short plates sit just below the notch at the top of the green gasket.

13. Place the electrophoresis module into the tank and fill the buffer chambers with 1x running buffer: 200 mL in the inner buffer chamber, 550 mL (1–2 gels) in the outer buffer chamber.

14. Load samples and run the gels using the running conditions appropriate to your application. Stop the run when the dye front reaches the reference line imprinted on the bottoms of the cassettes.

15. After electrophoresis is complete, turn off the power supply and disconnect the electrical leads.

16. Remove the lid from the tank and remove the gels from the cell. Pour off and discard the running buffer.

17. To open the cassette, align the arrow on the opening lever with the arrows marked on the cassette and insert the lever between the cassette plates at indicated locations. Apply downward pressure to break each seal. Do not twist the lever.

18. Pull the two plates apart from the top of the cassette, and gently remove the gel.

Straining

1. Briefly rinse the gel with double distilled water.

2. Shake the gel in 0.2M imidazole for 5-10 minutes.

3. Discard the imidazole solution.

4. Shake the gel in 0.3M ZnCl2 for 30 seconds.

5. Discard the zinc solutions and wash the gel with double-distilled H2O.

6. Visualize the gel against a dark surface.

Protein Recovery

1. Add 1 mL of elution buffer.

2. Crush the gel piece.

3. Centrifuge at 5,000 −10,000×g for 10 minutes.

Downstream Analysis

1. Transfer 1 mL of 50mg/L Pd(II) solution from the reaction to a 1.5 mL tube.

2. Centrifuge at 1,2000 r.p.m. for 30 minutes.

3. Transfer the solution t−o the cuvette. Do not touch the wall of the 1.5 mL tube.

4. Add 2 mL of DI water to the cuvette.

5. Measure the absorbance of the solution.

6. Determine the concentration of K2PdCl6 according to the calibration curve.

7. Calculate the original concentration of undiluted solution.

Team:HK SSC/Experiments