Team:XMU-China/Experiments
● Ni-bead with EpCAM coated
● 5% PBS-BSA buffer (see Appendices)
● Antibody of EpCAM (PE labeled)
● Flow Cytometer (BD FACSVerseTM)
Prepare 5% PBS-BSA: Weigh 0.5 g BSA, then add 1×PBS to the volume being 10 mL, then mix it evenly to make sure the solid dissolved.
1. Make distributions:
Add reagent to 3 1.5 mL tubes respectively according to the following instructions. Add PBS-BSA first then the antibody.
1) Sample Group: 5 μL EpCAM-Ni bead + 185 μL PBS-BSA + 10 μL antibody of EpCAM.
2) Positive Control: 5 μL EpCAM-Ni bead + 185 μL PBS-BSA + 10 μL antibody of EpCAM.
3) Negative Control: 5 μL EpCAM-Ni bead + 195 μL PBS-BSA.
2. Wash the free antibody:
Take out the filter of a 10 μL filter tip and insert it into a 200 μL tip to make a 200 μL filter tip.
The above three tubes are respectively subjected to the following operations:
1) Transfer the 200 μL of solution to the 200 μL filter tip, then pipette it up and down to mix evenly.
2) Use a syringe to squeeze out the solution in the 200 μL filter tip.
3) Remove the filter tip and place it in the previous tube.
4) Rinse the tip with 450 μL of 1×PBS buffer.
5) Take out the tip, then transfer 150μL of buffer from 4) to the filter tip and pipette up and down to mix evenly.
6) Use a syringe to squeeze out the solution in the 200 μL filter tip.
7) Repeat 5) to 6) three times, at which time 450 μL of the solution from 4) was filtered.
8) Use a pair of scissors to cut the thin end of the filter's head and loosen the filter.
9) Transfer 200 μL of 1×PBS buffer to a new 1.5 ml tube.
10) Use a pipette to insert the cut filter tip into the tube from 9) and pipette it up and down for several times.
3. Flow cytometer test:
Precautions:
1) Turn on the regulator first, then turn on the computer, and finally turn on the instrument 30 minutes before measuring.
2) Check if the sheath tank has a solution, if the waste container is full, and if the dehumidifier is turned on.
3) Remember to perform a two-step self-test.
4) Please use daily clean after measuring.
5) Shutdown sequence: Click shut down on the software, turn off the instrument, then turn off the computer, and finally turn off the regulator.
● SYL3C-FAM (Sangon Biotech®, see Appendices)
● EpCAM (ACROBiosystems®)
● Binding Buffer (see Appendices)
● Fluorescence Spectrometer (Shimadzu® RF-6000)
● Polarizer (from a pair of 3D glasses)
1. Make distributions in 1.5 mL centrifuge tubes:
1) Tube 1 (Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-FAM + 179 μL binding buffer.
2) Tube 2 (Aptamer-EpCAM Group): 1 μL 100 μM (100 pmol) SYL3C-FAM + 179 μL binding buffer.
2. Sample processing:
The above 2 tubes are simultaneously subjected to the following steps:
1) Denature at 95℃ for 10 minutes.
2) Remove the tubes, then put them at room temperature for about 20 minutes.
3) Add 20 μL of 100 μg/mL EpCAM protein to Tube 2.
4) Add 20 μL of binding buffer to Tube 1.
5) Incubate all tubes in a shaker for 40 minutes (37℃, 200 rpm).
Note: Avoiding light in whole process is necessary.
3. Measure fluorescence intensity from vertical/parallel direction:
1) Turn on the instrument for 30 minutes before measuring.
2) Choose "Emission Mode". The excitation wave length is 495 nm, and the range of emission wave length is from 500 nm to 600 nm.
3) Before measuring, dilute the solution from step 2 to about 3 mL with binding buffer in the fluorescence cuvette.
4) Attach a polarizer to the exit of the excitation light, and attach another polarizer to the receiving inlet of the emission light to make sure the polarization directions of the two polarizers are vertical/parallel.
5) Start measuring to obtain I∥, max (Aptamer), I⊥, max (Aptamer), I∥, max (Aptamer + EpCAM), I⊥, max (Aptamer + EpCAM).
● Sequence of SYL3C-FAM:
5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-FAM-3'
● Binding Buffer (pH 7.3~7.5) contains:
1×PBS buffer with 5 mM MgCl2
● SYL3C-TEG-biotin (BoruiTM, see Appendices)
● C3-FITC (BoruiTM, see Appendices)
● C4-FITC (BoruiTM, see Appendices)
● Binding Buffer (see Appendices)
● 50×TAE concentrate solution (Solarbio®)
● Agarose (Biowest®)
● DNA dye (TransGen® GelStain)
● Distilled water
● Microwave oven
● 10×Loading buffer (Takara®)
● Electrophoresis instrument
Dilute 50×TAE concentrate solution to 1×TAE buffer with distilled water.
1. Make distributions in 1.5 mL centrifuge tubes according to the following:
1) Tube 1 (Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-TEG-biotin + 19 μL binding buffer.
2) Tube 2 (C4 Group): 1 μL 100 μM (100 pmol) C4-FITC + 19 μL binding buffer.
3) Tube 3 (C3 Group): 1 μL 100 μM (100 pmol) C3-FITC + 19 μL binding buffer.
4) Tube 4 (C4&Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-TEG-biotin +1 μL 100 μM (100 pmol) C4-FITC + 18 μL binding buffer.
5) Tube 5 (C3&Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-TEG-biotin +1 μL 100 μM (100 pmol) C3-FITC + 18 μL binding buffer.
Note: The Tube 2, 3, 4 and 5 should be coated by tin foil properly after distributing.
2. Sample processing:
The above 5 tubes are simultaneously subjected to the following steps:
1) Denature at 95℃ for 10 minutes.
2) Anneal to room temperature for 20 minutes.
3. Prepare 3% agarose gel:
1) Weigh 0.9 g of agarose in a 100 mL Erlenmeyer flask.
2) Add 30 mL of 1×TAE buffer into the flask from 1).
3) Make agarose melt by microwave oven (medium-high heat, about 3 minutes).
4) Add 3 μL of GelStain, mix by shocking.
5) Assemble gel pouring apparatus by inserting gate into slots.
6) Pour agarose gel into the gel tray.
7) Cool for 40 minutes to solidify the DNA agarose gel.
8) Remove the pouring apparatus, put the gel into an electrophoresis instrument.
4. Electrophoresis:
1) Add 2 μL of 10×Loading buffer to each tube, then pipette up and down to evenly mix.
2) Pipette all samples which have been mixed with loading buffer into the slots.
3) Turn on the electrophoresis instrument, set the working electric current at 150 mA and the working electric voltage at 110 V.
4) Electrophoresis for 50 minutes.
5. Gel imaging:
Turn off the instrument, take the gel into the gel formatter to take and save photos.
● Sequence of SYL3C-TEG-biotin:
5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-TEG-Biotin-3'
● Sequence of C3-FITC:
5'-CTCTGTAGTGTTTTTTTTTTTTTT-FITC-3'
● Sequence of C4-FITC:
5'-TCTGTAGTGTTTTTTTTTTTTTTT-FITC-3'
● Binding Buffer (pH 7.3~7.5)contains:
1×PBS buffer with 5 mM MgCl2
● SYL3C-TEG-biotin (BoruiTM, see Appendices)
● C3-FITC (BoruiTM, see Appendices)
● EpCAM (ACROBiosystems®)
● Magnetic Beads (InvitrogenTM)
● Binding & Washing Buffer (see Appendices)
● Incubation Buffer (see Appendices)
● Vortex (IKA® lab dancer)
● Fluorescence Spectrometer (Shimadzu® RF-6000)
1.8 μL of 100 μM SYL3C-TEG-biotin is diluted by 1×B&W buffer up to 75 μL, and 1.2 μL of C3-FITC is also diluted by 1×B&W up to 50 μL.
The tube of diluted C3-FITC should be coated properly by tin foil in order to avoid being quenched.
1. Make distributions in 1.5 mL centrifuge tubes according to the following:
Tube 1 (Blank Control): 100 μL incubation buffer.
Tube 2 (Negative Control): 25 μL diluted SYL3C-TEG-biotin + 25 μL 1×B&W buffer.
Tube 3 (Competition Group): 25 μL diluted SYL3C-TEG-biotin + 25 μL diluted C3-FITC.
Tube 4 (Competition Group without EpCAM): 25 μL diluted SYL3C-TEG-biotin + 25 μL diluted C3-FITC.
Tube 5 (Positive Control): 0.6 μL 100 μM C3-FITC + 100 μL incubation buffer.
Note: The Tube 3, 4 and 5 should be coated by tin foil properly after distributing.
2. Denature, anneal and renature:
The Tube 2, 3, and 4 are denatured at 95°C for 5 minutes, then anneal to room temperature.
3. Wash magnetic beads:
1) Resuspend the beads and vortex for 10 seconds to make them even.
2) Transfer 75 μL of 10 mg/mL beads to 3 200 μL centrifuge tubes (ie 25 μL per tube).
3) Add 25 μL of 2×B&W buffer to each tube.
4) Vortex for 5 seconds to resuspend.
5) Place the tubes in a magnetic field for 1 minute, then aspirate the supernatant.
6) Remove the tubes from the magnetic field and add 50 μL of 1×B&W buffer to each tube.
7) Repeat steps 4) to 5).
8) Add 50 μL of 1×B&W buffer to each tube.
4. Bind to magnetic beads:
1) Add 50 μL of solution of Tube 2,3 and 4 from step 2 to 50 μL of washed beads, respectively.
2) Vortex for 5 seconds to resuspend.
3) Incubate at room temperature for 30 minutes in the dark.
4) Vortex for 5 seconds to resuspend.
5) Place the tubes in a magnetic field for 2 minute, then aspirate the supernatant.
6) Remove the tubes from the magnetic field and add 100 μL of 1×B&W buffer to each tube.
7) Repeat steps 4) to 5).
8) Add 100 μL of incubation buffer to Tube2, 3 and 4, respectively.
5. Compete:
1) Add 17 μL of 100 μg/mL EpCAM to Tube 2 and 3. And add 17 μL of incubation buffer to the last each tube.
2) Incubate at room temperature for 40 minutes in the dark.
3) Place the tubes in a magnetic field for 2~3 minutes, then pipette 100 μL of the supernatant into a new 200 μL centrifuge tube and mark it.
4) Measure the fluorescence intensity of Tube 1, 5 and the supernatant from 3).
Note: Before measuring, the solution of each tube will be further diluted by adding 3, 000 μL of incubation buffer into it. The excitation wave length is 495 nm, and the range of emission wave length is from 500 nm to 600 nm.
● Sequence of SYL3C-TEG-biotin:
5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-TEG-Biotin-3'
● Sequence of C3-FITC:
5'-CTCTGTAGTGTTTTTTTTTTTTTT-FITC-3'
● 2×Binding & Washing Buffer (pH 7.5) contains:
10 mM Tris 1 mM EDTA 2 M NaCl
● Incubation Buffer (pH 7.5) contains:
20 mM Tris 140 mM NaCl 5 mM KCl 1 mM MgCl2
● Cas12a (Alt-R® CRISPR-Cas12a (Cpf1) System)
● crRNA (IDT®)
● TE Buffer (Sangon Biotech®)
● DNaseAlertTM Substrate Nuclease Detection System (IDT®, see Appendices)
● 21nt ssDNA (BoruiTM, see Appendices)
● Black 96-well plate (Corning®)
● Microplate reader (Tecan Infinite® M200 Pro)
● Add 26.7 μL of filtered TE buffer into the tube of crRNA to make a 75 μM stock.
● Add 88.6 μL of filtered TE buffer into the tube of ssDNA to make a 100 μM stock.
1. Form the RNP complex:
1) Prepare 3 200 μL tubes. Add 1 μL of Cas12a and 1 μL of diluted crRNA into each nuclease-free 200 μL tube.
2) Incubate Cas12a with crRNA at 37℃ for 30 minutes to form the RNP complex.
2. Pretreatment of substrate:
1) Prepare 3 DNaseAlertTM Substrate single-use tubes. Add 5 μL of Nuclease-Free Water to each tube.
2) Add 5 μL of 10×DNaseAlertTM Buffer to each tube.
3. Make distributions:
1) Cas12a Group: DNaseAlertTM Substrate (from step 2) + 2 μL RNP complex (from step 1) + 1 μL diluted ssDNA + 37 μL filtered TE buffer + 40 μL Nuclease-Free Water.
2) Negative Control: DNaseAlertTM Substrate (from step 2) + 40 μL Nuclease-Free Water + 40 μL Nuclease-Free Water.
3) Positive Control: DNaseAlertTM Substrate (from step 2) + 1 μL DNase I + 39 μL filtered TE buffer + 40 μL Nuclease-Free Water.
4. Measure fluorescence intensity by microplate reader:
1) Incubate the reaction system at 37℃ for 30 minutes, then transform the reaction system into the black 96-well plate.
2) Before measure, add 12 μL of TE buffer into each tube.
3) Set temperature: 37℃, choose plate type: Black. Set excitation wavelength: 536 nm and emission wavelength: 556 nm. Choose mode: Optimal.
4) Measure fluorescence intensity at 30 min, 40 min, 50 min, 60 min, 70 min, 90 min, 110 min (Timing is from the beginning of incubation).
● DNaseAlertTM Substrate Nuclease Detection System contains:
1) DNaseAlertTM Substrate (25 single-use tubes (50 pmol per tube) or 2 tubes bulk substrate (2 nmol per tube))
2) DNaseAlertTM Buffer (250 μL)
3) Nuclease-Free Water (2 mL)
4) DNase I Enzyme (25 μL)
5) Nuclease Decontamination Solution (50 mL)
● Sequence of 21nt ssDNA:
5'-CAGGCCAACCCCCCATGACAA-3'
● TE Buffer (Sangon Biotech®)
● DNaseAlertTM Substrate Nuclease Detection System (IDT®, see Appendices)
● Fluorescence spectrometer (Shimadzu® RF-6000)
1. Make distributions:
Add reagent into 2 DNaseAlertTM Substrate single-use tubes according to following instruction. Avoiding light is necessary during the whole process.
1) Negative Control: 5 μL Nuclease-Free Water + 5 μL 10×DNaseAlert Buffer + 40 μL Nuclease-Free Water + 40 μL Nuclease-Free Water.
2) Enzyme Group: 5 μL Nuclease-Free Water + 5 μL 10×DNaseAlert Buffer + 39 μL Nuclease-Free Water + 40 μL Nuclease-Free Water.
Place the two tubes at room temperature for 30 minutes to enough dissolve fluorescent substance.
2. Make distributions further according to the time gradient:
1) Prepare 6 200μL-PCR tubes in advance, then divide into 2 groups (3 tubes per group). Mark "W" and "I" on the tubes of two groups, respectively. Further mark the tubes according to the time gradient of 3 min, 15 min and 30 min (e.g. "W 3", "I 15").
2) Add 1 μL of DNase I into the DNaseAlertTM Substrate single-use tube of Enzyme Group.
3) Mix immediately, then transfer 30 μL of reaction liquid from each single-use tube to each PCR tube marked before. Place all PCR tubes in 37℃ water bath. Then start timing.
4) Each tube: React for corresponding time, then measure florescence intensity immediately.
3. Measure fluorescence intensity:
1) Turn on the instrument for 30 minutes before measuring.
2) Choose "Emission Mode". The excitation wave length is 536 nm, and the range of emission wave length is from 540 nm to 580 nm.
3) Before measuring, dilute the reaction liquid to about 3 mL with TE buffer in the fluorescence cuvette.