1. Pick one clone from LB plate stored at 4 ℃.
2. Inoculate the clone in 2 mL LB broth, 37℃, 220 rpm, overnight.
3. Inoculate the overnight culture at 1:100 ratio to 100 mL LB.
4. Incubate at 37℃, 220 rpm, until OD(600)=0.6.
5. Chill the tubes on ice-water bath for 15 min.
6. (Pre-cool the centrifuge) Centrifuge at 4℃, 4000 xg, centrifuge for 15 min to collect the bacteria, and discard the supernatant.
7. Gently resuspend pellets in 100 mL pre-cold Double-Distilled Water. Stay on ice for 5 min.
8. Centrifuge at 4℃, 4000 xg, centrifuge for 10 min to collect the bacteria, and discard the supernatant.
9. Gently resuspend pellets in 50 mL pre-cold Double-Distilled Water. Stay on ice for 5 min.
10. Centrifuge at 4℃, 4000 xg, centrifuge for 10 min to collect the bacteria, and discard the supernatant.
11. Gently resuspend pellets in 2 mL pre-cold 10% glycerol. Stay on ice for 5 min
12. Centrifuge at 4℃, 4000 xg, centrifuge for 10 min to collect the bacteria, and discard the supernatant.
13. Gently resuspend pellets in 50 μL pre-cold 10% glycerol.
14. Add 100 ng plasmid DNA. Stay on ice for 30 min.
15. Transfer the cells into the pre-cold cuvette.
16. Wipe moisture from the cuvette and insert the cuvette into the device, with the parameters below:

17. Immediately add 1 mL pre-warmed SOC medium.
18. Transfer the cells to 1.5 mL EP tube. Incubate at 37℃, 220 rpm, 1h.
19. Centrifuge at 5,000 rpm, 3 min. Resuspend the pellets in 100 μL LB.
20. Spread the cells on pre-warmed LB+Amp plates. Incubate at 37℃, overnight

1. Inoculate the bacteria in 1.5ml EP tubes and incubate for more than 12 hours to make sure that the concentration of bacteria tends to be stable
2. Prepare multiple (the number is decided by how many time points you want) 10ml tubes and liquid medium, each tube is inoculated with 100uL bacterial solution from Step 1, and incubation.
3. Put the tube on the ice once it reaches the set time.
4. Measure its OD600 with spectrophotometer
5. Measure its CFUs:
   1. Dilute the solution with different magnifications
   2. Choose 3 appropriation dilution multiples and plate 100ul bacteria solution onto solid culture plates (“appropriate” means each plate has 30-300 colonies) Incubated them at 37°C
   3. Count the colony's number and determine the original CFUs
6. Repeat Step 4-5 for all the tubes.

1. Add an appropriate amount of agarose and TAE to the beaker. The ratio of agarose mass to TAE volume is shown in the following figure.

2. Bring slurry to a boil in a microwave (heat about 2 minutes on high power).
3. After the agarose solution is slowly cooled (but still liquid), add GELRED at a ratio of 1:20000.
4. Set up the gel tray in the gel box (in the casting orientation, which is perpendicular to the long axis of the gel box) and insert a comb. Pour molten agarose into the gel box. The agar is completely set when it becomes opaque (< 20 min).
5. Add the loading buffer/dye to the DNA samples to a final concentration of 1×.
6. Load samples and marker on the gel.
7. Run the gel at constant voltage of 120~150V. When the gel has run far enough (the bromophenol blue should still be in the gel), view the gel using the gel documentary.

1. Plate Coating
   To achieve optimal sensory efficiency, glycerol bacteria or other preserved bacterial strains must be coated on LB plates and cultured overnight.
2. Vaccination
   Place freshly cultured bacterial strains on a weighing plate, and perform subsequent operations in a clean bench. Dip the tip of the tweezers in 70% alcohol and slightly burn it on an alcohol lamp to sterilize the tip. Use the tweezers to pick up a sterile plastic tip or toothpick, select a monoclonal colony from the plate, and then place the plastic tip or toothpick dipped in the bacterial strain into a bacterial culture tube containing 3 mL of LB broth. This operation can also be performed using an inoculation loop.
3. Cultivation
   Cultivate the bacteria overnight at 37℃ and 200 rpm, controlling the cultivation time to approximately 16 hours, not exceeding 18 hours.
4. Re-inoculation Culture
   Inoculate fresh overnight bacteria into a fresh culture in a 1:500 ratio based on the amount of supercompetent bacteria required. For instance, add 100 μL of fresh overnight bacteria to 50 mL of the supercompetent bacterial culture medium provided in the kit. Cultivate at 37°C and 200 rpm for approximately 2.5-3.5 hours until the OD600 reaches 0.6-0.75.
5. Preparation of Competent Bacteria
   1. When the OD600 of the cultured bacteria reaches 0.6-0.75, place the bacterial culture in an ice bath and cool for 15 minutes. Note: All subsequent operations must be performed at 4°C or in an ice bath.
   2. Centrifuge at 4000 g at 4°C for 5 minutes to collect bacteria, and discard the supernatant. Note: The centrifuge must be pre-cooled.
   3. If the initial bacterial culture volume is 50 mL, follow the subsequent procedures. Adjust volumes proportionally for other volumes.
   4. Resuspend the bacterial pellet gently using 20 mL of pre-cooled supercompetent preparation reagent. Suspend very lightly and slowly to avoid disrupting the bacteria.
   5. Ice bath for 15 minutes.
   6. Centrifuge at 4000 g at 4°C for 5 minutes to collect bacteria, and discard the supernatant.
   7. Gently resuspend the bacterial pellet using 2 mL of pre-cooled supercompetent preparation reagent B. Suspend very lightly and slowly to avoid disrupting the bacteria.
   8. Ice bath for 15 minutes.
   9. On an ice bath, divide the suspension into 50-200 μL aliquots as needed.
   10. Use immediately or freeze in liquid nitrogen or an ethanol dry ice bath and store at -70°C. Efficiency typically remains stable for up to 6 months.
6. Use of Competent Bacteria
   1. For Determining Competent Bacteria Efficiency or Converting Ligation Products:
      1. Slowly melt the competent bacteria on ice (freshly prepared competent bacteria can be used directly). To test the efficiency of competent bacteria, add 10 pg-1 ng plasmid, but the volume of plasmid should not exceed 10% of the volume of competent bacteria. For converting ligation products, add 2-10 μL of ligation product for every 50 μL of competent bacteria.
      2. Gently flick the centrifuge tube with your fingers to mix the bacteria and plasmids or ligation products. Incubate in an ice bath or ice water bath for 30 minutes.
      3. Heat shock in a 42°C water bath for 2 minutes.
      4. Immediately after heat shock, place in an ice water bath for 2 minutes.
      5. Add 900 μL of LB broth and incubate at 37°C and 200 rpm for 1 hour.
      6. For efficiency testing, plate 100 μL onto LB plates containing the appropriate antibiotics. For ligation products, centrifuge at 2000-3000 g for 1 minute or longer to allow the bacteria to fully precipitate. Remove approximately 900-950 μL of LB culture medium. Resuspend the bacterial pellet in the remaining LB culture medium and plate onto LB plates containing the appropriate antibiotics. Incubate overnight at 37°C.
   2. For Plasmid Transformation:
      1. Dissolve the competent bacteria and add at least 50 ng of plasmid to 30-50 μL of competent bacteria. The amount of plasmid can be 1 μg or more, but the volume should not exceed 10% of the volume of competent bacteria.
      2. Gently flick the centrifuge tube with your fingers to mix the bacteria and plasmid.
      3. Incubate in an ice bath or ice water bath for 10 minutes.
      4. Plate all of the bacterial suspension directly onto LB plates containing the appropriate antibiotics and incubate overnight at 37°C.

1. Pick one clone from LB plate stored at 4 ℃.
2. Inoculate the clone in 2 mL LB broth, 37℃, 220 rpm, overnight.
3. Inoculate the overnight culture at 1:100 ratio to 100 mL LB.
4. Incubate at 37℃, 220 rpm, until OD(600)=0.6.
5. Chill the tubes on ice-water bath for 15 min.
6. (Pre-cool the centrifuge) Centrifuge at 4℃, 4000 xg, centrifuge for 15 min to collect the bacteria, and discard the supernatant.
7. Gently resuspend pellets in 100 mL pre-cold Double-Distilled Water. Stay on ice for 5 min.
8. Centrifuge at 4℃, 4000 xg, centrifuge for 10 min to collect the bacteria, and discard the supernatant.
9. Gently resuspend pellets in 50 mL pre-cold Double-Distilled Water. Stay on ice for 5 min.
10. Centrifuge at 4℃, 4000 xg, centrifuge for 10 min to collect the bacteria, and discard the supernatant.
11. Gently resuspend pellets in 2 mL pre-cold 10% glycerol. Stay on ice for 5 min
12. Centrifuge at 4℃, 4000 xg, centrifuge for 10 min to collect the bacteria, and discard the supernatant.
13. Gently resuspend pellets in 50 μL pre-cold 10% glycerol.
14. Add 100 ng plasmid DNA. Stay on ice for 30 min.
15. Transfer the cells into the pre-cold cuvette.
16. Wipe moisture from the cuvette and insert the cuvette into the device, with the parameters below:

17. Immediately add 1 mL pre-warmed SOC medium.
18. Transfer the cells to 1.5 mL EP tube. Incubate at 37℃, 220 rpm, 1h.
19. Centrifuge at 5,000 rpm, 3 min. Resuspend the pellets in 100 μL LB.
20. Spread the cells on pre-warmed LB+Amp plates. Incubate at 37℃, overnight

1. Dilute the overnight culture with medium provided by the kit with ratio of 1:500, and cultivate at 37℃, 200 rpm. After 2.5-3.5 hours, OD600 can reach 0.6-0.7.
2. Put the bacterial fluid on ice and cool it for 15 min. (The following steps should all be on ice)
3. (Pre-cool the centrifuge) 4℃, 4000 xg, centrifuge for 5 min, discard the supernatant.
4. Resuspend the bacteria pellet with 20 mL Reagent A softly and slowly.
5. Ice bath for 15 min.
6. (Pre-cool the centrifuge) 4℃, 4000 xg, centrifuge for 5 min to collect the bacteria, and discard the supernatant.
7. Use 2 mL pre-cooled Reagent B to resuspend bacteria pellet softly and slowly.
8. Ice bath for 15 min.
9. Package on ice, 50-200 μL per tube according to the need.
10. (For storage) Freeze with liquid nitrogen or ethanol in a dry ice bath and keep at -80℃.

1. Confirm the objective.
2. Verify the dilution buffer and blocking buffer.
3. Calculate dilution and set up your layout according to the protein extracted and your objective.

1. Digest and collect cells and use PBS to wash 2~3 times
2. Add Ripa into cell pellet and resuspend, put it on ice for 1 h
3. Add loading dye (dilute from 4× to 1×)
4. 95℃ 5~10min

1. Select an appropriate SDS-PAGE gel. (15% or 12%)
2. Loading equal mass of protein in a inverted order (loading the ladder lastly to make up with wrong loading)
3. Run the gel (Use lower voltage (80mV) at first to make a line when running gel. Then increase the voltage to higher, like 120mV. After finishing running and before getting out the gel, set a low voltage(30mV) in case of diffusion.) Prepare cooling water when running gel!(for semi-dry method)

1. Soak the membrane in methanol, if using a PVDF membrane. Then in a balance buffer (semi-dry method needs to use balanced buffer. Methanol is necessary)
2. Assemble the SDS-PAGE gel and the membrane in the transfer cassette. Heavy band on the bottom of the transfer cassette (higher temperature and ion concentration)
3. Run the transfer
4. Cooling it in water

1. Prepare blocking buffer and antibody dilution buffer: dissolve 5% BSA into 1×TBST
2. Place the membrane in a container and cover with blocking buffer. For chemiluminescent western blot, incubate the membrane with gentle rocking overnight at 4 °C, or for 1 h at room temperature
3. Cover the membrane with diluted primary antibody (Incubate with gentle rocking overnight at 4 °C, or 1~2h at room temperature.)
4. Wash the membrane three times with 1×TBST, 10mins each
5. Cover the membrane with second antibody (1h at RT)
6. Wash the membrane three times with 1×TBST, 10mins each

1. Clean the imaging scanning bed (clean it every time between different membranes to detect!)
2. Cover the membrane with ECL.
3. Adjust the mode (chemiluminescent/ colormetric) and exposure time and number of photos
4. After scanning, merge and rename photos. Export photos.

Wash down the ECL with TBST and put in -20℃

1. PVDF is very easy to dry
2. Add secondary antibody next to the fridge.

1. Culture at least 10mL LB broth with Carbenicillin(50 μ g/mL)from glycerol bacteria, 37℃ 220 rpm, for no more than 12h.
2. Add 10ml cultured bacteria into 1L LB broth with Carbenicillin(50 μ g/mL), then grow in the shaker under 37℃220 rpm, meansure the OD600 by the Ultraviolet spectrophotometer, until the OD600 reaches 0.6-0.8.
3. Add 10ml ultrapure water dissolved-20% Arabinose solution (mass fraction), then incubate at 30℃ in the shaker with 220 rpm.

1. After 20h, collect the bacteria by centrifuging at 6,000g, 15min, 4℃.
2. Discard the supernatant and resuspend the pellet by 80ml pre-cooled TBS buffer in total. Add PMSF solution, which is dissolved in 100% ethanol, to final concentration at 0.8mM.
3. Sonic the TBS-bacteria solution under 600W, work 5s following stop 3s, 30min total.
4. Centrifuge the soniced solution uner 14,000g for 45min, 4℃. Collect the supernatant.

1. Add 2mL Ni-charged resin into the supernatant. Incubate in 4℃ on Rotating mixing instrument, overnight.
2. Use gravity column to flow through, and wash by TBS with 40mM immidazole for 20mL. Elute by TBS with 300mM immidazole for 12mL.
3. Use a 10kDa concentration tube to concentrate 5mL of it to 500μ L.
4. Load the concentrated solution to chromatographic protein purifier.

1. Mix the samples and 4×NuPAGE loading buffer together, respectively.
2. Boil the mixture on heating block at 98℃ for at least 15min. Then put into a pre-cooled centrigue, 6,000g, 5min at 4℃.
3. Load the samples into pre-cast 4-20% gels, run in MOPS buffer, constant voltage at 125V, for 60min.
4. Take the gel out and stain with Comassie blue.

1. Run the SDS-PAGE gel as discribed previously.
2. Transfrom the brand to 0.45μm PVDF membrane by the Genescript®eblot L1.
3. Block the membrane in 5% TBST-dissolve BSA, for at least 1h under room temperature.
4. Transform the membrane to the HRP-linked His tag antibody solution, which is 1:2000 diluted in 1×TBST. Incubate overnight at 4℃ on 40 rpm horizontal shaker.
5. Wash the membrane with TBST for 3 times, 15min each.
6. Use enhanced chemiluminescence to detect the brands.

1. 100 μL 2ng/ml of HER2 standard is added to each well.
2. Incubate 120 min at RT on shaker, 160 rpm.
3. 100 μL of purified anti-HER2 scFv per each well (10 and 100 ng/μL) was bound to the HER2 antigen.
4. Incubating at 37 °C for 60 min.
5. Wash 5 times with 100 μL/well wash buffer.
6. Add His-tag-HRP（MC10）Mouse Monoclonal Antibody (LABLEAD H2104).
7. Incubate 120 min at RT on shaker, 160 rpm.
8. Wash 5 times with 100 μL/well wash buffer.
9. Add 100 μL/well TMB substrate, and incubate at room temperature in the dark for 20 minutes.
10. Add 100 μL/well stop solution.
11. Within 30 minutes, perform a dual-wavelength measurement using a microplate reader to determine the OD values at the maximum absorption wavelength of 450 nm and the reference wavelength of 570 nm or 630 nm. The calibrated OD value is the measurement at 450 nm minus the measurement at 570 nm or 630 nm.

1. Inoculate the overnight bacteria solution 100ul into 10mL medium, static in 37°C for needed culturing time (usually 5-6h)
2. Harvest Bacteria to the needed concentration: the volume should be less than bacteria medium 1:cell medium 100, and the number should be cell number : bacteria CFUs at ratio 1:25 to 1:50
   1. Centrifuge at 4000g for 10 minutes, or 12000rpm for 2 minutes for 1.5mL EP tube
   2. Resuspend in appropriate volume of PBS
   3. (optional) Dye the bacteria and wash off the excess dye
3. Inoculate bacteria in the cell. Incubate at 37°C, 5%CO2 for an appropriate time
4. Observe under microscope, do FACS or other experiment

1. Label on all the tubes and dishes: date + kind + passage number + other required things.
2. Different flasks/culture dishes/wells have different capacities of culture medium.
3. Always add things including cells against the wall of the container (unless you need to mix in culture dish).
4. Always count cells and observe cells to determine its confluence and vitality before adding mixture.
5. Always add culture medium and rinse before adding cells.
6. The condition of cell incubation and passage can be found on ATCC.
7. Be gentle when opening and closing the incubation box to prevent cell uneven growth. Don’t put your dish on the edge of the box to prevent it from dropping when you reach the dishes deep inside.
8. Discard pipette under right hand instead of above the dish.
9. Put your dish cover far away from operating area. Don’t operate above dish and dish cover. The best method is to open a crack with left hand.
10. When triturating, don’t let all liquid go out of pipette to prevent bubbles.

• Medium: 90% DMEM +10% FBS +1% Antibiotics
• Subcultivation Ratio: 1:5 to 1:10
• Medium Renewal: Depend on the daily observation.

• Medium: 90% McCoy's 5A +10% FBS +1% Antibiotics
• Subcultivation Ratio: 1:2 to 1:4
• Medium Renewal: 3-4 Days
• Notice:
  • The cell's adhesion ability is quite weak. Few floating cells were widely observed even after weeks of culturing, especially after thawing or subculturing.
  • The culturing in the first week needs to be very careful. Do not disturb the cells too often. To avoid cell loss, all the medium should not be discarded before centrifuge in the first week.

• Medium: See the protocol "Media Recipes For MCF10A Cells"
• Subcultivation Ratio: 1:3, no more than 1:4
• Medium Renewal: 3-6 Days
• Notice:
  • The adhesion process of the cell is quite slow, do not disturb it for at least 3 hours after subculturing.
  • The adhesion force of the cell is quite strong, usually requiring more than 10 minutes at 37°C.
  • The cell comes from an epithelial cell, tending to form large pieces of tissue. The cell density is easily underestimated.
  • The medium component of the cell is relatively complex, some components might influence the measurement and experiment results.
  • The cells will die out soon under low cell density. Control the subculturing ratio.

1. Gentle agitation in 37°C water bath in 2 min to thaw.
2. Remove from the water bath as soon as thawed.
3. Decontaminate with 75% ethanol.
   Operation starting from here should be under strict aseptic conditions.
4. Centrifuge with 5.0 mL medium, 500g, 3-5 min.
5. Discard the supernatant.
6. Resuspend in medium and dispense into a 10 cm² dish.
7. Incubation.

1. Remove the medium.
2. Rinse the cell layer with PBS to wash off the trypsin inhibitor.
3. Add 1.0 mL 0.25% Trypsin-0.53 mM EDTA. Wait until dispersed (usually 2 min).
   Notice: Do not shake when digesting or there will be clumping.
4. Add 1.0 mL serum-contained medium or soybean trypsin inhibitors to stop digesting.
5. (Optional) Centrifuge at an appropriate speed and discard the supernatant, then resuspend in the appropriate volume of the medium.
6. Transfer cells into new culture vessels in the appropriate ratio.
7. Incubation.

1. Prepare the cryoprotectant, reference recipe: 60% Complete growth medium + 30% FBS + 10% DMSO.
2. Do Steps 1-5 in Subculturing, resuspend in 2 mL appropriate cryoprotectant instead of medium in step 5.
3. Put the cryopreservation tube in a cell-freezing container, then put the container at -80°C.