Construction of target plasmid

1、 PCR (System: 25ul - Small EP Tube)

（1） Experimental principle
1. Polymerase chain reaction (PCR) refers to the process of amplifying
complementary sub chain DNA to the mother chain template DNA in vitro, catalyzed
by DNA polymerase, using the mother chain DNA as a template and specific primers
as extension starting points. Through continuous steps such as denaturation,
annealing, and extension, PCR is a DNA synthesis amplification technique.

2. PCR buffer: General composition: KCl, Tris Cl (room temperature pH 8.3),

MgCl2.
① KCl: promotes primer annealing, and high concentrations of KCl inhibit Taq DNA
polymerase activity;
② Tris Cl: Adjust the pH value to make the reaction system more alkaline;
③ MgCl2: regulates Taq DNA polymerase activity, affects primer annealing,
specificity of PCR products, etc

3. Primer: Two artificially synthesized oligonucleotide sequences that determine the

specificity of PCR.
1）The basic principles of primer design:
① Primer length: 18-24 bp, must be a certain length within a certain range to ensure
sequence uniqueness and reduce the possibility of the sequence existing at non target
sequence sites; The upper limit of primer length is mainly related to reaction
efficiency, so it generally cannot be too long, as being too long will cause its
extension temperature to exceed 72 ℃, which is the optimal temperature for Taq
enzyme.
② Product length: ordinary PCR 200-500bp; Real time fluorescence quantitative PCR
50-150bp.
③ The balance and GC content of primers: The base composition in primers should be
randomly distributed as much as possible to avoid the accumulation of purine or
pyrimidine bases; The proportion of (G+C) in effective primers is 40-60%, and too
high (non-specific amplification) or too low (reduced specificity) is not conducive to
triggering the reaction; The GC content of upstream and downstream primers
should not differ too much.
④ Primer dissolution temperature (Tm value): The Tm value of primers is generally
controlled between 55-60 degrees, ensuring that the Tm values of upstream and
downstream primers are consistent as much as possible, generally not exceeding 2
degrees. If the G+C content in the primer is relatively low, the primer length can be
made slightly longer while ensuring a certain annealing temperature.
⑤ Primers themselves: Complementary, dimeric, or hairpin structures at the 3 'end

1
between primers may also lead to PCR reaction failure; The several bases at the 3
'end of the primer must be strictly paired with the template DNA and preferably have
a low stability structure; The 5 'end sequence has less impact on PCR than the 3' end
sequence, and is commonly used to introduce modification sites or markers.

2）The process of primer design:

① Software: Prime Premier
Process: Copy template sequence → file → new → DNA sequence → paste → OK
→ S (forward primer) → Edit: Hairpin (hairpin structure), Dimer (D-dimer), False
priming: Starting from the 3 'end, delete, click Analyze → prime → observe Rating
(greater than 70), GC content (40% -60%) → copy primer sequence
② Software: Gene snap
Process: Primers → Add Primer → Paste the copied primer sequence from the
previous software → Pull 15-20 bp forward (forward primer) or backward (backward
primer)

（2） Experimental steps

1）system
Addition
amount
① Template DNA 50-100ng
Forward
1ul
② primer
Primers: Backwar
1ul
d primer
③ Enzyme 0.5-1ul
④dNTP 2ul
⑤5 x buffer 5ul
Suppleme
⑥ddH2O
nt to 25ul

2）Amplification process
① Pre denaturation: 94 (or 95) ℃, 5 minutes
② Denaturation: 94 ℃, 10s
③ Annealing: (Tm value of forward primer+backward primer)/2-5), 15 seconds

④ Extension: 68 ℃, 0.06 x length

⑤ Renaturation: 68 ℃, 5 minutes

3）Agarose gel electrophoresis identification:

2
① Larger fragments 10%:
30ml 1xTAE small piece glue+0.3g agarose powder+3ul green nucleic acid dye
Large block adhesive 60ml 1xTAE+0.6g agarose powder+6ul green nucleic acid dye
② 15% for smaller fragments
③ Electrophoresis: 120V constant voltage, HRS 25 minutes.

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2、 Overlapping PCR
1）Calculation: V1/V2=C2m1/c1m2
2）system
Addition amount
① Template DNA Fragment 1 50-100ng
Fragment 2 50-100ng
② Primers: Forward primer for 1ul
fragment 1
Backward primer for 1ul
fragment 2
③ Enzyme 0.5-1ul
④dNTP 2ul
⑤5 x buffer 5ul
⑥ddH2O Supplement to 25ul

3）Amplification process
One step method:
① Pre denaturation: 94 (or 95) ℃, 5 minutes
② Denaturation: 94 ℃, 10s
③ Annealing: (Tm value of forward primer+backward primer)/2-5), 15 seconds
④ Extension: 68 ℃, 0.06 x length
⑤ Renaturation: 68 ℃, 5 minutes
Two step method:
Step 1 (without primers):
Pre denaturing at 95 ℃ for 5 minutes
Denaturation: 95 ℃, 10s
Annealing: Tm value of overlapping fragments -5,15s
Extension: 68 ℃, extension segment length X0.06
10 cycles
Step 2 (Add Primers and Centrifuge):
Pre denaturing at 95 ℃ for 5 minutes
Denaturation: 95 ℃, 10s
Annealing: (Tm value of forward primer+backward primer)/2-5), 15s (if overlap
is not possible, lower annealing temperature: 45, 50, 55, 60)
Extension: 68 ℃, target fragment length X0.06

4）Agarose gel electrophoresis identification

4
3、 Seamless cloning (system: 20ul --1.5ml EP tube)
一．Experimental principle
1. Basic principle: A method of homologous recombination based on insertion fragments and
linearized vector end 15-20 bp homologous sequences to achieve cloning.
2. Experimental process:
1) The fragment to be inserted is obtained by designing specific primers (with a 15-20 bp
homologous arm between the primer and the vector enzyme cleavage site) for PCR amplification.
If multiple fragments are inserted, they need to be separately PCR and then overlapped PCR to
connect them.
2) Linearization of Carrier:
System (20ul)
Template 3ug (3000ng)
Enzyme ①: EcoR I 1ul
Enzyme 2: MIu I 1ul
Cut Smart 2ul
Add ddH2O to 20ul
Incubation: 37 ℃, 3h
electrophoresis
Rubber recycling
Measure concentration
3) Seamless cloning:
system
① Target fragment volume=0.04 x length/concentration
② Carrier volume=0.02 x length/concentration
③ Seamless cloning of 5x buffer 4ul
④ Seamless cloning enzyme 2ul
⑤ Add ddH2O to 20ul
plenti Plenti-v2
① Target fragment ① GRNA primer volume=6ul (diluted 100 times
volume=0.04 x after mixing F+R)
length/concentration ② Carrier volume=0.02 x length/concentration
② Carrier volume=0.02 x ③ Seamless cloning of 5x buffer 4ul
length/concentration ④ Seamless cloning enzyme 2ul
③ Seamless cloning of 5x buffer ⑤ Add ddH2O to 20ul
4ul
④ Seamless cloning enzyme 2ul
⑤ Add ddH2O to 20ul
Incubation: 37 ℃, 30min

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4、 Slow rotation
1. Take out the competent cells (Escherichia coli cells) from the -80 ℃ freezer (the
first box in the middle of the second layer below) and let it melt on ice for a few
minutes. Attention: Handle with care.
2. 100ul of competent cells+10ul of seamlessly cloned product from the previous
step, cannot be blown!
3. Ice bath for 30 minutes: Sensory cells are prepared by the CaCl2 method.
Escherichia coli cells expand into spherical shapes in a 0 ℃ CaCl2 hypotonic
solution, transforming the DNA in the mixture to form anti DNase hydroxyl calcium
phosphate complexes that adhere to the cell surface, promoting cell absorption of
DNA complexes, and restoring spherical cells for division and proliferation/// The
binding of Ca2+to DNA neutralizes the negative charge of DNA, and the binding of
Ca2+to bacterial cell membrane neutralizes the charge, overcoming the negative
charge repulsion between foreign DNA and bacterial cell membrane. Ca2+forms
coordination complexes with phosphates of DNA and bacterial cell membrane
lipopolysaccharides, promoting the binding of DNA and lipopolysaccharides.
4. Heat shock: 42 ℃, 45s. Heat shock increases the temperature, releases lipids
from the cell membrane, forms pores on the cell membrane, and allows DNA to enter
the interior of bacteria.
5. Ice bath for 5 minutes: The temperature decreases, protein is released from the
cell membrane, the proportion of lipids increases, the fluidity of the cell membrane
increases, and the pores on the cell membrane disappear.
6. Add 500ul LB.
7. Shaking table: 37 ℃, 1h. Plasmids form closed loops and replicate.
8. Centrifuge: 5000rpm, 5 minutes.
9. Discard 500ul of supernatant and mix well.
10.Laying: Add 10-15 beads to the AMP board, add the suspension onto the beads,
shake and mix well.
11.Incubator at 37 ℃: 12-14h.

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5、 Plasmid Xiaoti
（1） Preparation before the experiment
1）Picking bacteria: Add a single colony on an AMP plate (with a pipette tip inserted)
to 9ml LB (15ml centrifuge tube).
2）Add 9ul of ampicillin (1:1000 ratio) to the centrifuge tube.
3）Place the centrifuge tube in a 37 ℃ incubator and shake overnight (14 hours).
（二）Plasmid extraction
1）Column balance: 200 ul Buffer CBS, centrifuged at 12000rpm for 1 minute.
2）Centrifuge: 15000g, 3 minutes, discard the supernatant after centrifugation, invert
the centrifuge tube (to prevent liquid reflux)/ice!
3）Remove residual water droplets (including water on the lid).
4）Add 250 ul Solution I (on ice) (containing 25 mM Tris HCl (pH 8.0), 10 mM
EDTA, 50 mM glucose) and resuspend. Transfer to a new 1.5ml EP tube and shake
well. Attention: RNase A is usually added to solution 1 before use. RNase A has a clear
function of degrading RNA in the solution. Due to the fact that RNase is a protein with poor
protein stability, solution 1 containing RNase A needs to be stored at a low temperature of 4
℃.
5）Add 2 50ul Solution II (containing NaOH, SDS), gently flip up and down 6-8
times and mix well to form an egg smooth liquid. Attention: The time should not exceed
5 minutes, and the centrifuge tube should not be subjected to severe vibration. Excessive time
and severe vibrations can cause sodium hydroxide to damage genomic DNA, and the broken
genomic DNA fragments will be extracted together with plasmids of similar size,
contaminating the sample.
6）Add 350ul Solution III (ice bath: acid-base neutralization will release heat)
(containing 3M potassium acetate and 5M acetic acid), gently flip up and down 8-10
times, mix well, and let it stand at room temperature for 2-5 minutes. The main function
of solution 3 is to neutralize the strong base added in the second step and remove impurities
such as proteins from the cells.
7）Centrifuge: 12000rpm, 10 minutes.
8）Suck the supernatant onto the adsorption column (be careful not to suck the
precipitate) and let it sit for a few minutes.
9）Centrifuge: 12000rpm, 1 minute, discard the waste liquid in the collection tube.
10）Add 500ul of W1 Solution to the column, centrifuge at 12000rpm for 1 minute,
and discard the waste liquid in the collection tube.
11）Add 500ul of Wash Solution to the column (pay attention to whether ethanol is
added), centrifuge at 12000rpm for 1 minute, and discard the waste liquid in the
collection tube. Repeat this step once.
12）Leave empty for 2 minutes, 12000rpm, and leave at room temperature with the lid
open for a few minutes (15-20 minutes).
13）Transfer the adsorption column to a new 1.5ml EP tube, add 50-100ul Elution
Buffer, and let it stand at 37 ℃ for 2 minutes.
14）Centrifuge: 12000rpm, 2 minutes, the target solution is in the EP tube.

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15）Measure concentration

16）Enzyme digestion identification

① Enzyme digestion system
Plasmid 500g/concentration (ug/ml) (ul)
Enzyme 1ul
Cut smart 2ul
Add ddH2O to 20ul
Incubate at 37 ℃ for 1 hour (30 minutes)
② Agarose gel electrophoresis
Comb: 1.0mm
Marker:gene Ruler:6ul

六、Quick turn
Preparation before the experiment: icing
1.20ul competent cells+1ug (1000ng) plasmid
2. Ice bath for 5 minutes
1. 42 ℃ heat shock for 90 seconds
2. Ice bath for 5 minutes
3. Laying: Use a gun to mark Zone 4
4. Incubate in a 37 ℃ incubator for about 14 hours
5. The next day, select bacteria: prepare 500ml LB, sterilize under high pressure
(120 ℃, 30min) → add 500ul AMP (1:1000) → select individual colonies → culture
on a shaker at 37 ℃ for about 14 hours

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七、Plasmid extraction
1. Centrifuge the shaken bacterial solution: 3000g, 20min, 4 ℃
2. Discard the supernatant, add 10ml Buffer P1 (with RNaseA added) to resuspend
the precipitate, and transfer it to a clean 50ml centrifuge tube. Vortex and mix well
3. Add 10ml Buffer P2 to lyse the bacteria, slowly invert several times until the
bacterial solution turns uniform blue, and let it stand at room temperature for 5
minutes
4. Add 10ml of Buffer P3 to neutralize the pH, slowly invert and mix until the blue
color is no longer visible, and the entire tube of bacterial solution turns into a uniform
white color
5. Centrifuge: 12000rpm, 5 minutes
6. Install the QIA filter Cartridge Cap onto the QIA filter Cartridge and place it in a
clean 50ml centrifuge tube using a mounting bracket for future use
7. Transfer the supernatant to QIA filter Cartridge and let it stand at room
temperature for 10 minutes
8. Gently inject the bacterial solution into a 50ml centrifuge tube (approximately
20ml) using a syringe, and add 2.5ml Buffer ER (endotoxin removal) to the filtrate.
Invert and mix several times, then ice bath for 30 minutes
9. Equilibrate the QIAGEN-Tip500 nucleic acid adsorption column with 10ml QBT
Buffer in advance for 5-7 minutes, and wait for QBT to filter until no liquid droplets
are dropped
10.Wash the impurities on the column with 30ml QC Buffer and repeat the wash once
11.Place the nucleic acid adsorption column in another clean 50ml centrifuge tube
and wash the plasmid with 15ml QN Buffer
12.Add 0.75 times the volume (10.5ml) of isopropanol, mix well, centrifuge (15000g,
30min, 4 ℃)
13.Discard the supernatant, add 75% ethanol, centrifuge (15000g, 10min, 4 ℃)
14.Discard the supernatant, absorb the liquid around the extraction block, and let it
stand for 5 minutes
15.Dissolve 100-200ul TE, blow and mix as gently as possible
16.Measure the concentration (x ug/ml) and adjust it to 1000ug/ml for transfection
into cells
The required additional TE amount (ul)=x ug/ml × v (already added TE amount ul) ÷
1000-v (already added TE amount ul)

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八、Plasmid extraction
1. Bacterial picking: ① Prepare 250ml LB liquid culture medium, sterilize under high
pressure at 121 ℃ for 30 minutes; ② Add 250ul AMPA to the culture medium, select
a single colony and inject the pipette tip into the culture medium together;
2. Shake culture: Shake at 37 ℃ for 14h-16h;
3. Prepare alcohol and high-pressure gun tips, centrifuge the bacterial solution in a
250ml centrifuge tube, wash thoroughly before use, and label properly; RNaseA
and lyseblue should be added to the P1 buffer, and P1 and P3 should be placed on ice;
4. Centrifuge: Centrifuge the shaken bacterial solution at 3000g, 20min, and 4 ℃.
Discard the supernatant and invert it. Use a negative pressure suction device to extract
the residual liquid;
5. Add P1: Resuspend 4ml of P1 buffer (oscillator) and transfer to a 15ml centrifuge
tube;
6. Add P2: Add 4ml of P2 buffer (NaOH, SDS, break the wall) and gently invert and
mix 4-6 times (turning into a uniform blue color). Leave it for 5 minutes (if left for
too long, the plasmid will also lyse and produce mixed bands);
7. Add P3: Add 4ml of P3 buffer, gently invert until completely white, incubate on
ice for 15 minutes;
8. Centrifuge (twice): 15000g, 30min, 4 ℃ centrifugation (pre cooling); Transfer
the supernatant to a new tube and centrifuge for 10-15 minutes;
9. After assembling the column, add 4 ml of QBT buffer (providing an alkaline
environment to allow DNA binding) to balance the column; Add the supernatant to
the column and let it flow freely;
10.Wash twice: Wash twice with 10ml QC;
11.Dissolve: Wash DNA with 5ml QF into a new 15ml centrifuge tube;
12.Isopropanol precipitation: Mix 3.5ml of isopropanol (0.7 times the volume),
5000g, 30min, centrifuge at 4 ℃, discard the supernatant (selectively precipitate
DNA, large molecule rRNA, mRNA);
13.Ethanol removal of isopropanol: Add 2ml of 70% ethanol, 15000g, for 10
minutes, and discard the supernatant (clean by suction);
14.Natural drying, add 50ul TE to dissolve;
15.Measure the concentration and adjust the concentration (1000ng/ul).

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cell culture
1、 Cultivation of adherent cells
1. Recovery
① Pre open the 37 ℃ water bath;
② Take a 15ml sterile centrifuge tube and add about 8ml of PBS;
③ Remove the frozen adherent cells from the liquid nitrogen tank and rapidly
shake them in a 37 ℃ water bath to completely melt the cells within 1 minute;
④ Transfer the melted cells to a 15ml centrifuge tube in ② and centrifuge at
1500rpm for 5 minutes;
⑤ Use a negative pressure suction device to remove the supernatant from the
centrifuge tube, and add 1ml of DMEM complete culture medium to resuspend the
cell pellet;
⑥ Add the cells to a 10cm cell culture dish and add 9ml of complete culture
medium;
⑦ Gently shake the culture dish up, down, left, and right to mix the cells,
observe the cell state and density under a microscope, and incubate in a 37 ℃, 5% CO2
incubator.

2. passage
① Take out a 10cm dish from the incubator and observe the cell density (4x)
and growth status (10x) under a microscope. If the density reaches 80-90% and the
condition is good, passage can be carried out;
② Take out DMEM complete culture medium, PBS, and trypsin from the 4
℃ refrigerator and place them in a super clean workbench for reheating; Place two
15ml sterile centrifuge tubes and passaging culture dishes on an ultra clean
workbench;
③ Use a negative pressure suction device to remove the culture supernatant
from the culture dish, add 2ml PBS along the dish wall, gently shake the dish to wash
the cells, and remove the supernatant;
④ Repeat the previous step;
⑤ Add 0.5ml of trypsin and PBS to a centrifuge tube, mix well, and then add to
a culture dish. Shake the dish to ensure that trypsin fully covers the cell surface, and
digest at 37 ℃ for 2-4 minutes;
⑥ Take out the cells and observe the digestion under a microscope. If the cell
shape becomes round and the intercellular space expands, it indicates complete
digestion. Immediately add 2ml of complete culture medium to terminate digestion;
⑦ Gently blow the cells with a pipette to completely detach them, and transfer
them into a 15ml sterile centrifuge tube. Centrifuge at 1500rpm for 5 minutes;
⑧ Use a negative pressure suction device to remove the supernatant from the
centrifuge tube, add 1ml of DMEM complete culture medium to resuspend the

11
precipitate (or directly add 11ml or 21ml of culture medium mixed well and then add
to a 10cm or 15cm dish);
⑨ Prepare new 10cm or 15cm culture dishes, add 10ml or 20ml DMEM
complete culture medium to each dish, evenly distribute the cell suspension into the
new dish, gently shake up, down, left, and right to mix the cells, observe the cell
status and density under a microscope, and culture in a 37 ℃, 5% CO2 incubator.

3. Freeze storage
① Add fresh isopropanol to the freezing box until the mark is reached, and
place it at 4 ℃;
② Remove cells from the incubator and observe their growth status and density
under a microscope. If they are in good condition and have a density of 80-90%, they
can be frozen;
③ After digesting the cells, transfer them to a 15ml centrifuge tube and
centrifuge at 1500rpm for 5 minutes;
④ Use a negative pressure suction device to aspirate the supernatant, add 1ml
of cell cryopreservation solution to resuspend the cell pellet;
⑤ Take 10ul of cell suspension and add it to a 96 well flat plate. Then add 90ul
of trypan blue to dilute it tenfold. After mixing well, take 10ul and wash it in a bovine
abalone counting plate. Count under a microscope;
⑥ Adjust the cell density to 10M/ml, gently blow and mix well, and evenly
distribute into each cryovial;
⑦ Mark the information on the cryotube, including cell name, name, and
packaging time, seal it with a sealing film, transfer it to a 4 ℃ pre cooled cryobox,
and then place it in a -80 ℃ freezer for gradient cooling and freezing;
⑧ The next day, transfer the cells to a liquid nitrogen tank for long-term
storage.

二、Cultivation of suspended cells

1. Recovery
① Pre open the 37 ℃ water bath;
② Take a 15ml sterile centrifuge tube and add about 3ml of RPMI-1640 complete
culture medium;
③ Remove the frozen suspended cells from the liquid nitrogen tank and rapidly shake
them in a 37 ℃ water bath to completely melt the cells within 1 minute;
④ Transfer the melted cells to a 15ml centrifuge tube in ② and centrifuge at 1500rpm
for 5 minutes;
⑤ Use a negative pressure suction device to remove the supernatant from the
centrifuge tube, and add 1ml of RPMI-1640 complete culture medium to resuspend
the cell pellet;

12
⑥ Add the cells to T25 cell culture flasks and add 6ml of complete culture medium;
⑦ Gently shake the culture bottle to mix the cells, observe the cell state and density
under a microscope, and place them upright in a 37 ℃, 5% CO2 incubator for
cultivation.

2. Passage
① Take out a 10cm dish from the incubator and observe the cell density (4x) and
growth status (10x) under a microscope. If the density reaches 80-90% and the
condition is good, passage can be carried out;
② Take out DMEM complete culture medium, PBS, and trypsin from the 4 ℃
refrigerator and place them in a super clean workbench for reheating; Place two 15ml
sterile centrifuge tubes and passaging culture dishes on an ultra clean workbench;
③ Use a negative pressure suction device to remove the culture supernatant from the
culture dish, add 2ml PBS along the dish wall, gently shake the dish to wash the cells,
and remove the supernatant;
④ Repeat the previous step;
⑤ Add 0.5ml of trypsin and PBS to a centrifuge tube, mix well, and then add to a
culture dish. Shake the dish to ensure that trypsin fully covers the cell surface, and
digest at 37 ℃ for 2-4 minutes;
⑥ Take out the cells and observe the digestion under a microscope. If the cell shape
becomes round and the intercellular space expands, it indicates complete digestion.
Immediately add 2ml of complete culture medium to terminate digestion;
⑦ Gently blow the cells with a pipette to completely detach them, and transfer them
into a 15ml sterile centrifuge tube. Centrifuge at 1500rpm for 5 minutes;
⑧ Use a negative pressure suction device to remove the supernatant from the
centrifuge tube, add 1ml of DMEM complete culture medium to resuspend the
precipitate (or directly add 11ml or 21ml of culture medium mixed well and then add
to a 10cm or 15cm dish);
⑨ Prepare new 10cm or 15cm culture dishes, add 10ml or 20ml DMEM complete
culture medium to each dish, evenly distribute the cell suspension into the new dish,
gently shake up, down, left, and right to mix the cells, observe the cell status and
density under a microscope, and culture in a 37 ℃, 5% CO2 incubator.

3. Freeze storage
① Add fresh isopropanol to the freezing box until the mark is reached, and place it at
4 ℃;
② Remove cells from the incubator and observe their growth status and density under
a microscope. If they are in good condition and have a density of 80-90%, they can be
frozen;
③ After digesting the cells, transfer them to a 15ml centrifuge tube and centrifuge at
1500rpm for 5 minutes;
④ Use a negative pressure suction device to aspirate the supernatant, add 1ml of cell
cryopreservation solution to resuspend the cell pellet;

13
⑤ Take 10ul of cell suspension and add it to a 96 well flat plate. Then add 90ul of
trypan blue to dilute it tenfold. After mixing well, take 10ul and wash it in a bovine
abalone counting plate. Count under a microscope;
⑥ Adjust the cell density to 10M/ml, gently blow and mix well, and evenly distribute
into each cryovial;
⑦ Mark the information on the cryotube, including cell name, name, and packaging
time, seal it with a sealing film, transfer it to a 4 ℃ pre cooled cryobox, and then
place it in a -80 ℃ freezer for gradient cooling and freezing;
⑧ The next day, transfer the cells to a liquid nitrogen tank for long-term storage.

14
 Preparation of lentivirus
一、Slow virus packaging
1. Pre warming of the culture medium
2. Take out the 293 T cell culture dish from the incubator and observe the cell density
under a microscope. If it reaches 70-80%, it can be packaged with lentivirus. (Key
steps)
3. 2-3 hours before transfection, replace the culture medium: Take a 15cm culture
dish into a biosafety cabinet and use a suction pump to discard the supernatant. Leave
a small amount of liquid to prevent it from drying out. Then, use a pipette and a
pipette to draw 20ml of culture medium (10ml at a time, draw 11 to 1ml). Tilt the
culture dish and slowly add it along the wall to prevent the adherent cells from being
scattered. After adding, put the culture dish back into the incubator for later use.
4. Configure the co transfection system and let it stand for 5 minutes each:
1) Plasmid tube: 500ul PBS+20ug plasmid (target plasmid+P1+P2+P3), let it stand
for 5 minutes.
15cm dish 10cm dish
Target plasmid 10.6ug 5.3ug
PLP1 (packaging plasmid) 5.3ug 2.65ug
PLP2 (packaging plasmid) 2.35ug 1.175ug
PLP3 (enveloped plasmid) 1.76ug 0.88ug
PBS 500ul 500ul

2) PEI tube: 500ul PBS+PEI, let it stand for 5 minutes.

15cm dish 10cm dish
PEI 60ul 30ul
PBS 500ul 500ul

3) Add system 2) into a 15ml centrifuge tube of system 1), mix and let stand for 20
minutes

5. Remove the culture dish and slowly add the co transfection system along the 4
corners of the dish, or tilt the dish and slowly drip it back into the incubator.
6. Virus collection: After 48 hours, collect the cell culture supernatant into a 50ml
centrifuge tube, seal it, and store it in a refrigerator at 4 ℃ for temporary storage. Add
20ml of fresh culture medium; Collect the second time after 72 hours and mix the two
supernatants.
7. Centrifuge (2500rpm, 15min), filter (0.45um filter membrane), seal and store in
the refrigerator for temporary storage, discard the culture dish.
二、Concentrated lentivirus

15
1. Overnight centrifugation: Centrifuge the collected virus supernatant for 14-16
hours (3000g, 10 ℃);
2. Pour the supernatant into another new centrifuge tube on the ultra clean bench,
invert it, and use a suction pump to dry the residual liquid (suction two or three times,
every five or six minutes);
3. Add an appropriate amount of resuspended solution (60ul/dish) and blow it once
or twice. Store it in a refrigerator at 4 ℃ for several hours and resuspend the virus in
a 1.5ml EP tube (resuspended solution: 581+1% HEPES);
4. Centrifuge the supernatant overnight and repeat the above steps (40ul/dish).
5. According to the situation, divide the virus into different volumes, label the virus
name and packaging time on the tube, and store at -80 ℃ for future use.
三、Determination of lentiviral titers
1. Transfect Jurkat cells
1) Take out Jurkat cells from a 37 ℃, 5% CO2 cell culture incubator and observe the
cell state under a microscope. If the cells are clear, plump, and in good condition, they
can be used for virus transduction;
2) Transfer Jurkat cells from T25 cell culture flasks to 15 mL centrifuge tubes and
centrifuge at 1500 rpm for 5 minutes at room temperature;
3) Discard the supernatant using a negative pressure suction device, add 1 mL of
RPMI-1640 complete culture medium for resuspension, and aspirate 10 μ L of the
supernatant
Cell suspension was diluted 10 fold with 90 μ L of trypan blue solution, and the cells
were counted under a microscope.
4) Take a 96 well flat bottom plate and add 0.5 million Jurkat cells to each well;
5) Add the concentrated virus into the wells at a decreasing dilution ratio, and
supplement with RPMI-1640 complete culture medium to 200 μ L. Add 0.1 μ L of
Polybrene (diluted 10 times and 1 μ L) to each well to promote transduction;
proportion 1：100 1：500 1：1000 1：2000
virus 2ul 4ul (diluted 10 2ul (diluted 10 1ul (diluted 10
times) times) times)
Cells 0.5 million 0.5 million 0.5 million 0.5 million
Polybrene 1ul 1ul 1ul 1ul
Tips: Set up negative control wells (without virus)
6) After sealing with sealing film, put it into PE gloves, centrifuge at 1200 g and 32
℃ for 90 minutes;
7) After centrifugation, disinfect the 96 well flat bottom plate and place it back in a 37
℃, 5% CO2 incubator for 4-6 hours of cultivation;
8) After 4-6 hours, remove the 96 well plate and transfer the corresponding cells into
a new 24 well cell culture plate,
And add 1 mL of RPMI-1640 complete culture medium;
9）Place the 24 well cell culture plate back in a 5% incubator at 37 ℃ and incubate
for 48 hours.

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2. Flow cytometry detection of Jurkat cell surface expression rate:
1) Configure FACS Buffer;
2) After 48 hours of Jurkat cell culture, take out a 24 well cell culture plate, gently
blow and mix the cells, aspirate 200 μ L and transfer it to a 96 well pointed bottom
plate;
3) Centrifuge at room temperature at 2500 rpm for 3 minutes;
4) Discard the cell supernatant, wash each well with 200 μ L FACS buffer, and
centrifuge at room temperature for 3 minutes at 2500 rpm;
5) Prepare antibody diluent according to the ratio, add 50 μ L of antibody diluent to
each test well, gently mix the cells, and incubate at 4 ℃ in the dark for 15 minutes;
6) Remove the plate and add 150 μ L FACS Buffer to each well to terminate
staining. Centrifuge at room temperature at 2500 rpm for 3 days
min；
10）Discard the supernatant, resuspend in 200 μ L FACS buffer, and detect the surface
expression rate of Jurkat cells on the machine.

3. Flow based result analysis:

Obtain the positive rate of virus transduction in Jurkat cells with different proportions.
Virus titer calculation:
Formula for calculating virus titer: T=(P * N)/(D * V)
T: Titer (TU/mL)
P=proportion of flow cytometry positive cells
N=number of transduced cells
D=dilution ratio
V=Transduction volume

MOI=T * v (virus volume)/number of cells per well (0.1 million, 0.1 X 106) →
V (required virus volume)
The required virus volume V=(MOI × cell count 0.1 X 106
)/virus titer

4、 Preparation of TRuC-T cells

1. T cell activation:

17
1) Prepare serum-free culture medium for T cells;
2) Extract 15 mL of venous blood from healthy individuals into EDTA-Na2
anticoagulant tubes;
3) Transfer the whole blood to a sterile 50 mL centrifuge tube in a super clean bench,
add an equal amount of D-PBS and mix thoroughly in a 1:1 ratio to dilute the whole
blood;
4) Take several 15 mL sterile centrifuge tubes, add Ficoll separation solution to the
bottom layer, slowly add diluted whole blood along the tube wall using a pipette and
spread it on top of Ficoll separation solution (Ficoll: diluted blood=1.2-1.5:2), pay
attention to the speed of whole blood addition, and do not mix diluted whole blood
with Ficoll separation solution;
5) 800 × g, centrifuge at 20 ℃ for 20 minutes, adjust the lifting speed to "0", and turn
off the centrifuge brake;
6) Carefully aspirate the white membrane layer between serum and Ficoll using a
sterile pipette into a centrifuge tube (avoiding suctioning Ficoll separation solution
as much as possible), and add D-PBS to fill it up to 1 cm below the scale line of the
test tube;
7) Centrifuge at room temperature for 5 minutes at 1500 rpm;
8) Use a negative pressure suction device to aspirate the supernatant, add 1 mL of D-
PBS to resuspend the precipitate, and then add D-PBS to make up to 1 cm below the
scale line of the test tube
9) Centrifuge at room temperature for 5 minutes at 1500 rpm;
10) Configure Buffer I solution: 10 mL D-PBS+2% AB serum (200ul);
11) Resuspend the cell pellet with 1mL Buffer I, transfer it to a sterile Ep tube,
aspirate 10 μ L, stain with trypan blue, and count using a abalone counting plate;
10) Flow cytometry was used to determine the proportion of CD3+T cells in PBMCs;
11) Add CD3/CD28 dynabeads (1 × 106 cells plus 10 μ L magnetic beads) in a ratio
of CD3+T cells: CD3/CD28 dynabeads=1:1, and rotate the shaker at 4 ℃ for 40
minutes to ensure full contact between the magnetic beads and the cells;
12) After 40 minutes, place the Ep tube containing cells on a magnetic rack for 1-2
minutes to allow the magnetic beads connecting T cells to adhere to the tube wall, and
carefully discard the supernatant;
13) Add an appropriate amount of serum-free culture medium for T cells, gently
mix magnetic beads and cells;
14) Spread cells at a density of 1 × 106 cells/mL (1M) onto a cell culture plate and
incubate in a 37 ℃ incubator.

2. T cell transduction:
1) Remove T cells activated for 24 hours (not exceeding 48 hours) from the
incubator;
2) Gently mix the cells with a pipette, take 10 μ L of cell suspension, dilute it 10 times
with trypan blue, and fill it into a abalone counting plate for counting;

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3) In a 96 well plate, T cells were added at a rate of 1 × 105/well, and viruses were
added according to different infection rates. 0.1 μ L of Polybrene transducer was
added to each well, and T cell culture medium was used to fill up to 200 μ L;
4) 1200 × g, centrifuge at 32 ℃ for 90 minutes;
5) Place the culture plate in a 37 ℃, 5% incubator for cultivation.
6) After 4 hours, remove the 96 well plate to the ultra clean table, use a pipette to
aspirate 100 μ L of culture medium from each well along the wall, replace with 150 μ
L of fresh T cell serum-free culture medium, and continue to culture at 37 ℃ in a 5%
incubator.

3. Flow cytometry detection of T cell surface expression rate:

1) Configure FACS Buffer (49mL D-PBS+1mL FBS+EDTA);
2) When T cells are cultured to a sufficient quantity, their surface positivity rate can
be detected. In a super clean bench,
Mix the cells in the well with a gun tip and aspirate an appropriate volume of cells to
the bottom plate of the 96 well tip;
3) Centrifuge at room temperature at 2500 rpm for 3 minutes;
4) Discard the cell supernatant, wash each well with 200 μ L FACS buffer, and
centrifuge at room temperature for 3 minutes at 2500 rpm;
5) Prepare flow cytometric antibodies according to the corresponding dilution ratio,
add 50 μ L of antibody to each well, mix gently, and incubate at 4 ℃
Incubate in the dark for 20 minutes;;
6) Remove the plate and add 150 μ L FACS Buffer to each well to terminate
staining. Centrifuge at room temperature at 2500 rpm for 3 days
min；
7) Discard the supernatant, add 200 μ L FACS Buffer and mix the cells. Centrifuge at
2500 rpm at room temperature for 3 minutes;
8) Repeat 7);
9）Discard the supernatant, resuspend the cells in 200 μ L FACS buffer, and detect T
cell transduction on the machine

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T cell in vitro functional experiment
一、In vitro killing experiment
1. Luciferase bioluminescence assay
1) Plate laying: After digestion of target cells, count them and lay them on a 96 well
white plate at 0.01 million/well. Fill with DMEM complete culture medium to 50
ul/well and incubate in a 37 ℃, 5% CO2 incubator;
2) T cell centrifugation, resuspension counting;
3) Calculation:

4）Add T cells according to the calculation in the previous step (with three wells set
for each effect target ratio), and set up a positive and negative control (negative
control group only added target cells and culture medium, positive control group only
added target cells and ddH2O). Incubate in a 37 ℃, 5% CO2 incubator: adherent cells
(20-24h)/suspended cells (5-8 h).
5）Take it out of the incubator and centrifuge at 2500rpm for 3 minutes.
6）Discard the supernatant, add 200ul of DMEM containing luc (1:200) to each well,
and incubate in the dark for 10 minutes.
7）On the machine, enzyme-linked immunosorbent assay (ELISA) reader.
8）Calculate the killing efficiency as (Max-V)/(Max Min) × 100%.

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2. RTCA method
1）Add 50ul DMEM complete culture medium into each hole of the E-Plate, put it
into the RTCA machine that has been started, and detect the background resistance
value of the Flat noodles (holes with cell index values less than 0.063 can be used for
subsequent steps);
2）Count the target cells after digestion and adjust the concentration to 0.2M;
3）Suspend the RTCA machine, take out the Flat noodles, add 50ul cell suspension
(i.e. 0.01million/hole) to each hole, and place it in the ultra clean workbench for
30min;
4）Put the Flat noodles back into the RTCA machine and click "Start" to test the
resistance value;
5）After the target cells adhere to the wall, centrifuge the T cells and perform
demagnetization counting;
6）Suspend the detection, take out the Flat noodles, add T cells of each group to the
hole in a ratio of 1:1 of the effect target ratio, and then fill it with DMEM complete
medium to 200ul/hole;
7）Put the Flat noodles back into the machine and click "Start" to continue the test.

二、FCM detection of T cell subtypes

1）After thoroughly blowing and mixing the T cells, extract 200ul to 1.5ml of EP
tubes and place them on a DynaMagTM-5 magnetic rack for demagnetization;
2）Suck out the cells to the 96 well pointed bottom plate,

21
 clinical
Pre experiment
一、Blood lifting
1. Whole blood: PBS=1:1
2. Ficoll: Diluted blood=6ml: 8ml.
3. Centrifuge: 800g, 25min, adjust the lifting speed to "0".
4. Absorb white membrane cells.
5. Centrifuge: 1700rpm, 7min, maximum speed adjustment for lifting and lowering.
6. Pour the supernatant into another centrifuge tube, and resuspend the precipitate in PBS.
7. Centrifuge: 1700rpm, 7 minutes.
8. Discard the supernatant, resuspend the precipitate in T cell culture medium, and incubate T25
in a supine manner for 24 hours.

二、activation
1. Count and add magnetic beads (1million plus 10ul).
2.4 ℃ rotational activation for 40 minutes.
3. Discard the supernatant and lay the board.

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