Bacterial Transformation

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1. Add 2.0 μL of DNA solution (purified) to Competent cells (Top10/BL21(DE3)) 50 μL (Please use from the “used” cells in our deep freezer after melting.)

2. Stand on ice 30 min

3. Heat 42℃for 45 s

4. Stand on ice for 2 min

5. Add 250 μL of SOC/LB medium

6. Incubate tubes at 37℃at 200 rpm for 1 hour

7. Plate50 μL/200 μL onto LK plates

8. Put the plate upside down and cultivate overnight at 37 °C

Restriction Enzyme Digestion

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Preparation of linearized vector fragments

1. Prepare reaction mixture as follows.

   Plasmid (about 0.1-1 μg/μL)	5 μL
   10X Cut Buffer	5 μL
   Restriction enzyme	0.4 μL
   dd H2O	39.6 μL
   Total reaction volume	50 μL

   *Note: Confirm the restriction enzyme units are enough to digest all plasmids. All operations should be done on ice. Don't forget to bring enzymes back to -25 °C after using. Please use the water bath to incubate the mixture overnight, but not heat block.

2. Please incubate the mixture at 37 °C overnight.

Plasmid DNA Mini Preparation

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1. Column Equilibrium Step: Add 500 μLof equilibration buffer BL to the adsorption column CP4 (place the column in a collection tube). Centrifuge at 12,000 rpm (~13,400×g) for 1 min, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. (Please use columns processed on the same day.)

2. Transfer 5–15 mLof overnight bacterial culture into a centrifuge tube and centrifuge at 5,000×g for 10 min. Invert the tube on paper for 5 min to remove as much supernatant as possible.

   *Note: If the bacterial culture volume is large, pellet the cells through multiple centrifugations and combine them into one tube. The amount of bacterial cells should be optimal for complete lysis. Excessive bacterial cells may lead to insufficient lysis and reduce plasmid extraction efficiency.

3. Add 500 μLof Solution P1 (ensure RNase A has been added) to the centrifuge tube containing the cell pellet. Re-suspend the bacterial cell pellet thoroughly using a pipette or vortex mixer. (1.5 ml EP tube)

   *Note: Ensure complete re-suspension of the bacterial pellet. Incomplete mixing may affect lysis, leading to lower yield and purity.

4. Add 500 μLof Solution P2 to the centrifuge tube and gently invert 6–8 times to ensure complete lysis.

   *Note: Mix gently—avoid vigorous shaking to prevent genomic DNA contamination. The lysate should become clear and viscous within no more than 5 min to avoid plasmid damage. If the solution remains cloudy, the bacterial load may be too high; reduce the amount for complete lysis.

5. Add 500 μLof Solution P4 to the centrifuge tube and immediately invert gently 6–8 times to mix thoroughly. A white flocculent precipitate will form. Let it stand at room temperature for ~10 min, then centrifuge at 12,000 rpm (~13,400×g) for 10 min. A pellet will form at the bottom of the tube.

   *Note: After adding P4, mix immediately to avoid localized precipitation. If small white precipitates remain in the supernatant, centrifuge again and collect the supernatant.

6. Transfer the supernatant from the previous step in batches to the filtration column CS (placed in a collection tube). Centrifuge at 12,000 rpm (~13,400×g) for 2 min, and collect the filtrate in a clean 2 mLcentrifuge tube (user-provided).

7. Add isopropanol (0.3× the filtrate volume, 400–500μL) to the filtrate. (Excessive isopropanol may cause RNA contamination.) Mix by inverting, then transfer the mixture to the adsorption column CP4 (placed in a collection tube).

   *Note: Due to potential filtrate loss, adjust the isopropanol volume accordingly. The CP4 column has a maximum capacity of 700 μL, so load the sample in batches.

8. Centrifuge at 12,000 rpm (~13,400×g) for 1 min at room temperature. Discard the flow-through and return the adsorption column to the collection tube.

   *Note: If the solution from Step 7 is loaded in batches, repeat this step for each aliquot.

9. Add 500 μLof protein removal buffer PD to the adsorption column CP4. Centrifuge at 12,000 rpm (~13,400×g) for 1 min, discard the flow-through, and return the column to the collection tube.

10. Add 600μL of wash buffer PW (ensure absolute ethanol has been added) to the adsorption column CP4. Centrifuge at 12,000 rpm (~13,400×g) for 1 min, discard the flow-through, and return the column to the collection tube.

    *Note: Letting the column sit for 2–5 min at room temperature after adding PW may improve impurity removal.

11. Repeat Step 10 with another 600μL of wash buffer PW. Centrifuge and discard the flow-through.

12. Reinsert the adsorption column CP4 into the collection tube and centrifuge at 12,000 rpm (~13,400×g) for 2 min to remove residual wash buffer.

    *Note: Ethanol residue may interfere with downstream enzymatic reactions (e.g., digestion, PCR). To ensure complete removal, air-dry the column at room temperature for a few minutes with the cap open.

13. Place the adsorption column CP4 in a clean centrifuge tube. Apply 50–300 μLof preheated 60 °C ddH₂O or elution buffer TB directly to the center of the membrane. Incubate at room temperature for 5 min, then centrifuge at 12,000 rpm (~13,400×g) for 1 min to collect the plasmid solution.

    *Note: For higher yield, reload the eluate onto the column and repeat Step 13. Elution efficiency depends on pH: Use elution buffers with pH 7.0–8.5 (pH <7.0 reduces yield). Minimum elution volume: 100 μL (lower volumes decrease recovery). Store DNA at –20℃ to prevent degradation. (Perform two elutions totaling 100 μL.)

PCR

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1. Please make two tubes of PCR mixtures as follows.

   Component	Volume
   ddH2O	(18.25 - x) μL
   10X PCR Buffer (with Mg²⁺)	2.5 μL
   dNTP (2.5 mM each)	2 μL
   Template DNA (final 50 ng/μL)	x μL
   Primer 1 (10 μM each)	1 μL
   Primer 2 (10 μM each)	1 μL
   Taq DNA Polymerase (2.5 U/μL)	0.25 μL
   Total	25 μL

2. PCR by following program PCR program

   1) 94 ℃ 3 min --- denature

   2) 94 ℃ 30 sec --- denature

   3) 55 ℃ 30 sec --- annealing

   4) 72 ℃ t min (min/kb) --- extension

   5) GOTO step 2), 30 times

   6) 72 ℃ 10 min

   7) 12 ℃ Forever

3. Check the PCR products by 1.0% agarose electrophoresis

Gel Electrophoresis

1. 1% or 1.5% Agarose gel electrophoresis, 100 V for checking (apply 12 μL/lane)

2. Use 5 μL/lane for Beyotime DNA Ladder (0.1-10 kb, 21 bands, figure below)

3. Stain the gel for 30 min with about 200 mL of GelRed (1/3300 diluted for bubble dyed)

4. Check the stained bands by Imaging system. Put the stained agarose gel on the UV tray.

5. Please recycle the GelRed solution in the light-protected boxes.

IPTG induction

Materials and Source

• Overnight culture
• LB broth with 50 μg/mL Kanamycin
• Refrigeration Centrifuge
• 100 mM IPTG

*Note: To prepare a 100 mM IPTG solution, 2.383 g of IPTG powder should be weighed and dissolved in 100 mL of deionized water. After filtration and sterilization, it should be packaged and stored at -20 ℃.

Procedure

1. The overnight cultures are diluted 100-fold in fresh LB medium with 50 μg/mL Kanamycin, and cultured at 37 ℃ until they reached an optical density (OD) at 600 nm of 0.6.
2. Cells could be pelleted at 4000 g and gently resuspended in an induction medium containing 0.6 mM, 0.8 mM, 1.2 mM IPTG and 50 μg/mL antibiotics. Protein expression is allowed to occur at 37 ℃ for 4 hours or 16 ℃ overnight.

*Note: 0.4 mM IPTG is not necessarily the optimal concentration. It is proper to make the concentration gradient, such as from 0.2 mM to 1.2 mM IPTG (0, 0.5, 1 mM first).

Nanodrop

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Materials used in the Nanodrop process

• Nanodrop instrument
• ddH₂O

• Liquid to be tested
• Solvent
• Delicate task wiper

Procedure

1. Before measuring the concentration of the substrate, first use 2 μL of ddH₂O in the cuvette of the instrument, and gently close and open the cover. Then use a delicate task wiper to wipe the cuvette.

2. Repeat the first step 2 times.
3. Use a pipette to drop 2 μL of the solvent, turn on Nano 2000c, click the blank button, and measure. Perform the zeroing procedure.

   *Note: If using ddH₂O (EB buffer) to elute the column which contains DNA, use ddH₂O (EB buffer) to measure the standard curve.

4. Use a pipette to drop 2 μL of the liquid to be tested on the cuvette, close the cover gently, and click the start button.
5. Write down the statistics of concentration, A260/280, and A260/230 on the screen and compare them with the expected data.
6. After finishing the Nanodrop process, use ddH₂O to wash the cuvette.

Streaking

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Procedure

1. Pick a well-separated colony on a plate with relatively few colonies. Using a heat-sterilised inoculating loop, streak the colony out onto a separate agar plate.
2. Aseptically remove a loopful of bacteria from the sample and transfer it to section 1 of the agar. Evenly distribute the inoculum over the surface of section 1 in a back-and-forth motion.
3. Flame sterilize the loop and let it cool.
4. Turn the plate and spread the bacteria from section 1 to section 2 as diagrammed.
5. Flame sterilize the loop again and repeat spreading the bacteria from section 2 to section 3.
6. Flame sterilize the loop again and repeat going into section 4 of the plate.

Mini-scale liquid culture in tubes

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1. Check the streaked colonies.
2. Bring LK medium from 4 ºC to room temperature.
3. Put 10 mL of LK medium into 50 mL tubes.
4. Pick up a colony with a pipette tip or inoculating loop and inoculate into the LK medium.
5. Please don’t forget to open the cap and seal with tape!
6. Shake at 37 ºC at 150–200 rpm for more than 12 hrs or overnight.
7. Transfer bacterial solution from 50 mL tubes to 2 mL Eppendorf tubes.

8. Centrifuge at 3000–5000 ×g for 10 min in SIGMA 1-14K centrifuge at room temperature.
9. Discard the supernatants to the bacteria drain.

Aim: various culture for mini-prep & growth curve

Preparation of LB medium for small culture

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Materials

1. 500 mL Bottle
2. LB premix powder (20 g for 1 L)

Procedure

1. Take 400 mL of MilliQ water into a cylinder.
2. Pour the water into the 500 mL bottle.
3. Weigh 8 g of premix LB powder and add it into the bottle.
4. Cover with aluminum foil and fix with sterilization tape or seal, then put into the autoclave.
5. Never forget to open the cap!
6. Autoclave for 20 min, at 121 ℃, under 2 atm pressure.
7. After cooling to 60 ℃, add 1/1000 volume of kanamycin (1 g/mL) before use.
8. Store at 4 ℃.

Preparation of LK plates

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Materials

1. 200–300 mL Flask
2. LB premix powder (LB nutrient agar)

Procedure

1. Measure 100 mL of MilliQ water using a cylinder.
2. Pour the water into a 200–300 mL flask.
3. Weigh LB nutrient agar for 100 mL and add into the flask.
4. Cover with aluminum foil and fix with sterilization tape or seal, then put into the autoclave.
5. If there is a cap, never forget to open it!

6. Autoclave for 20 min at 121 ℃, under 2 atm pressure.
7. After around 90 min, check the autoclave temperature. If below 60 ℃, move the bottles to the bench and add 1/1000 volume of kanamycin (1 g/mL). If there are bottles with agar, move them to a 60 ℃ dry heater. Ensure the kanamycin concentration is 50 μg/mL. Mix gently!
8. Pour 20 mL medium into each 9 cm dish.
9. Let the dishes stand until solidified. If space and time allow, keep lids open during solidification to prevent condensation on the lid.
10. Store the plates upside down at 4 ℃.

RIPA lysis

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Procedure

1. For 1 mL bacterial solution, centrifuge to remove the supernatant.
2. Wash the pellet once with PBS. Centrifuge (12,000 rpm, 5 min), remove the supernatant, and gently tap the tube bottom to disperse the pellet.
3. Add 200 μL lysis buffer (protease inhibitor optional). Vortex gently or tap to mix, then lyse on ice for 20 min, vortexing every 4 min.
4. Optional: For better lysis, treat bacteria with lysozyme before using this buffer.

Ultrasonication

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Materials

• Fisherbrand FB505 Ultrasonic Crusher
• Processing capacity: 0.2–1000 mL
• 500 W, 20 kHz
• Programmable, 1 s to 10 h
• Fisherbrand FB50 Ultrasonic Crusher
• Processing capacity: 0.2–50 mL
• 50 W, 20 kHz
• Manual operation

Procedure

1. Centrifuge the induced sample (3000 ×g, 5 min) and discard the supernatant.
2. Wash the cells 1–2 times with equal volume of PBS buffer, centrifuging each time.
3. Resuspend the pellet in 10 mL PBS buffer.
4. Insert the ultrasonic probe into the suspension (kept in an ice-water bath).

5. Ultrasonic conditions: 150 W, work for 1 s with a 3 s interval, total 20 min.
6. After sonication, centrifuge at 4 ℃, 12,000 ×g for 20 min.
7. The supernatant is the crude enzyme solution (take 40 μL as the supernatant sample).
8. Resuspend the pellet in 10 mL PBS buffer (take 40 μL as the pellet sample).
9. Store both the supernatant and pellet suspension at –20 ℃.

Bicinchoninic Acid Assay

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Equipment

• Microplate reader (capable of measuring absorbance at 562 nm)
• 96-well plate
• 37 ℃ incubator
• Adjustable pipettes and tips
• Deionized water
• EP tubes

Reagent Preparation

*Note: If crystals appear in BCA Reagent A, gently heat until dissolved.

1. 1× PBS: Dilute 5× PBS with deionized water to 1× before use. Store at 4 ℃ for up to 12 months.
2. BSA Standard (2 mg/mL): Dissolve 2 mg BSA in 1 mL 1× PBS. Aliquot and store at –20 ℃ to avoid freeze–thaw cycles.
3. BCA working solution: Mix BCA Reagent A and Reagent B at 50:1 immediately before use. Store at 4 ℃ for up to 24 h.

*Note: If crystals form in BCA Reagent A, gently heat at 37°C until fully dissolved. If flocculent precipitates appear, mix well before use.

Procedure

1. Preheat the microplate reader for ≥30 min and set the wavelength to 562 nm.

2. 96-well plate
   

3. Prepare 20 μL standard solutions, then add 20 μL samples to the 96-well plate. Add 200 μL BCA working solution to each well and mix thoroughly. Incubate at 37°C for 30 min, then measure the OD values at 562 nm.
   • Blank well (0 µg/mL): Record as $A_{empty}$
   • Standard wells: Record as $A_{standard}$
   • Sample wells: Record as $A_{sample}$
4. Calculate:

   • ΔA_standard = A_standard − A_empty
   • ΔA_sample = A_sample − A_empty

5. Data Analyse
   • Standard Curve: Plot ΔA(y-axis) vs. standard concentration (µg/mL, x-axis).

     standard

   • Sample Concentration: Substitute ΔAinto the standard curve equation to obtain x (µg/mL).

     sample

   *Tip: If samples were further diluted, multiply the result by the dilution factor (n).

Western Blotting

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SDS-PAGE Electrophoresis

1. Run the SDS-PAGE by constant 15 mA for stacking gel and 30 mA for separating, or constant 200Vfor whole gels.
2. When dye reached 5 mm in distance from the bottom of gel, stop running the PAGE.

Procedure

1. Immerse a new PVDF membrane in 100% methanol for 30 sec (you can recycle this methanol.)
2. Add the Wet transfer buffer to the PVDF membrane, and shake it for >15 min.
3. During shaking, immerse 2 filter papers in the transfer buffer together with the membrane.
4. Immediately after finishing gel, apart the gel plates from the device, open the glasses, and remove the stacking gel carefully and cut the bottom corner at the 1st lane.
5. Immerse the separating gel in transfer buffer and shake it for 15 min.
6. Wear transparent PE gloves and put a filter paper onto the black sponge on the black plastic side, and put the gel on the filter papers. Note, please construct the gel holder cassette in the large box with sufficient transfer buffers. Check bubbles, please remove them.
7. Put the PVDF on the gel, and put another filter paper on it.
8. Please push the gel from the surface of papers to remove bubbles.
9. Close the gel holder cassette and insert it to the electrode module.
10. Please put 2 frozen ice boxes in the buffer tank, and apply constant 100V for 60 min. Due to your results, you should refine suitable time for transferring (40-90 min).
11. Please prepare the blocking solution (2% BSA or 5% skim milk in PBS-T, and so on) during transferring.
12. Immediately after transferring, open the lid and mark the cutting corner of gel and the transferred side of PVDF membrane by a water-resistant ball pen.
13. Immerse the membrane into the blocking solution and shake it for 30-60 min.
14. Wash the membrane 1-3 times of 3-5 min shaking in the PBS-T.
15. Please dilute the 1st antibody by PBS-T (or blocking solution) to make 1-1.4 mL. In case of anti-FLAG, recommended dilution is 1/2,000 for the first trial. Note, please bring antibody on ice and after using, please bring antibody back to -25 ºC immediately!
16. Sand the membrane between parafilms or clear pockets to start 1st antibody reaction.
17. After 45-60 min, wash the membrane with the PBS-T, 3-5 times by 3-5 min shaking.
18. Please dilute the 2nd antibody by PBS-T to make 1-1.4 mL. In case of anti-mouse IgG, 20,000 times dilution should be recommended. Note, please bring antibody on ice and after using, please bring antibody back to -25 ºC immediately!
19. Add the diluted 2nd antibody (1-1.4 mL) to the PVDF membrane between parafilms.
20. After 45-60 min, wash the membrane with the PBS-T, 3-5 times by 3-5 min shaking.
21. Before finishing reaction, bring ECL solution A and B (Biosharp) and take each 0.5-0.7 mL and warm them at room temperature, and turn on the BioRad ChemiDoc MP.
22. Mix ECL A with B and incubate the mixture (total 1-1.4 mL) with the PVDF membrane on a parafilm or in a clear pocket for 1 min.
23. Take images of chemiluminescence from the membrane using BioRad ChemiDoc MP. Choose Application-Blot-Chemiluminescence. Put the membrane on the Blot/UV/StainFree tray.
24. To merge molecular markers, please choose Application-Blot-Colorimetric after obtaining the chemiluminescence image.

Crystal Violet Staining

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Procedure

The experiments were performed on a 96 well plate.

1. The initial E. coli stock culture was prepared by adding 1mLinoculum into 9mL standard LB broth and allowing the E. coli to grow overnight (16-18 hrs) at 37℃ (induced by IPTG whenunder orbital shaker at 200 rpm.
2. Next day, the optical density (OD) at 600nm was checked. Value at 600nm must be 2.0 or dilute it.
3. Few wells of plate are filled with 200μL of fresh LB as blank.
4. The culture was diluted in the ratio of 1:100 with 195μL of fresh LB broth and 5μL of culture in the well.
5. The plates were then transferred to thermostatic static incubators maintained at 37℃.
6. To avoid evaporation, the external area of the plate was filled with less amount of distilled water.
7. After 24 hrs, the 96 well plate was taken from the incubator and the planktonic cells were removed.
8. The wells were then washed using sterile PBS.
9. Each well was then added with 200 μLof 0.1% crystal violet and was kept for 15 min for staining.
10. The crystal violet was then removed and washed with sterile PBS and kept the plate for air drying for 5 min.
11. The biofilm was quantified in duplicate, after adding 200 μL of 95% ethanol, by an enzyme-linked immunosorbent assay plate reader at 570 nm.