Notebook

Engineering Cycle 1: A molecular classifier used to distinguish between bacterial or viral infections

Experimental materials:DNA fragments synthesized by Inc、target, weight probe, Bst polymerase、dNTPs

Experimental apparatus：PCR Amplifier、electrophoresis apparatus、quantitative real-time fluorescence detector

Electrophoresis verification of the weight probe construction

Experimental date: April 14th,2023

Experimental members: Gui Yuyin, Huang Jiong, Ouyang Weiyi

Probe synthesis

	track strand 10μM	F-code 10μM	Buffer 10μM	Water	60μL 250nM system
P1	1.5μL	1.5μL	6μL	51μL	√
P2	1.5μL	3μL	6μL	49.5μL	√
P3	1.5μL	4.5μL	6μL	48μL	√
P4	1.5μL	6μL	6μL	46.5μL	√
P5	1.5μL	7.5μL	6μL	45μL	√

After the configuration is complete, put it into the PCR instrument, select program YHX-90-1.td, reagent capacity of 60 μL, and start running.

The procedure for the temperature reduction is as follows：

Steps	Temperature	Time
1	90-85-80-75-70-65-60-55-50-45-40℃	One minute for each stage
2	37℃	5 Minutes
3	25℃	5 Minutes
4	4℃	forever

Electrophoresis verification

① Configure gel solution (15% electrophoresis gel)

4.25ml	ddH2O	10mL
3.75ml	40% acrylamide (Gel solution)	10mL
2ml	5×TBE	10mL
7μL	TEMED	Finally add it,after adding it, the glue stared to solidify
55μL	10%APS	Finally add it,after adding it, the glue stared to solidify

ps：Remember to pour the glue and insert a 10-well electrophoresis comb.

② Configure sample (while waiting for gel in 15 minutes)

1μL loadingbuffer	4 μL of the mixture of loadingbuffer and sample was added to the wells
5μL sample

Report Probes in response to weight molecular via the TMSD reaction

Experimental date: April 19th,2023

Experimental members: Yang Huixiao, Huang Jiong, Ouyang Weiyi

Synthesis the probe

Code-FAM 10μM	Code-BHQ1 10μM	Water	Buffer
3	3	39	5

After the configuration is complete, put into the PCR instrument, select program YHX-90-1.td, reagent capacity of 50 μL, and start running.

The temperature reduction procedure is as follows:

Steps	Temperature	Time
1	90-85-80-75-70-65-60-55-50-45-40℃	One minute for each stage
2	37℃	5 Minutes
3	25℃	5 Minutes
4	4℃	forever

Reaction system preparation

	Fluorescent reporter probe 600nM	weight molecular 500nM	Buffer 10x	ddH2O
0	10	0	5	35
10	10	1	5	34
20	10	2	5	33
30	10	3	5	32
40	10	4	5	31
50	10	5	5	30
60	10	6	5	29
70	10	7	5	28
80	10	8	5	27
90	10	9	5	26
100	10	10	5	25

After the fluorescence curve was stabilized, the fluorescence intensity depended linearly on the concentration of weight molecular, demonstrating that the fluorescence reporter probe was able to produce a linear response to different concentrations of weight molecular, enabling the quantitative conversion of weight molecular signal to fluorescence intensity signal.

Detection and weighting of the target signal by weight probes

Experimental date: Feb.23th,2023

Experimental members: Gui Yuyin, Zhou Mingzhen

Synthetic weight probes. (P1) and the fluorescent reporter probe. (P2)

Weight probe. (P1) Unit of μL

ARG1-track strand 5μM	Code（weight molecular） 5μM	Buffer 10x	Water
6	6	6	42

CD177-track strand 5μM	Code（weight molecular） 5μM	Buffer 10x	Water
6	24	6	24

VNN1-track strand 5μM	Code（weight molecular） 5μM	Buffer 10x	Water
6	30	6	18

Fluorescence reporter probe. (P2) Unit of μL

FAM-code 10μM	BHQ1-code 10μM	Buffer 10x	Water
3	3	6	48

Reaction system preparation

	P1 500nM	P2 500nM	target 50nM	bst	dNTP	Buffer 10x	ddH2O
ARG1(1,2)	1.5	6	2	1	1	5	33.5
CD177(4,2)	1.5	6	2	1	1	5	33.5
VNN1(5,2)	1.5	6	2	1	1	5	33.5
ARG1(1,5)	1.5	6	5	1	1	5	30.5
CD177(4,5)	1.5	6	5	1	1	5	30.5
VNN1(5,5)	1.5	6	5	1	1	5	30.5
ARG1(1,10)	1.5	6	10	1	1	5	25.5
CD177(4,10)	1.5	6	10	1	1	5	25.5
VNN1(5,10)	1.5	6	10	1	1	5	25.5

As shown in the figure above, after the fluorescence signal is stabilized, the solution fluorescence intensity is linear with the target weight and the target concentration. The weight probe can detect and weighting the target signal.

Fluorescent validation of the weight addition

Experimental date: Feb.27th,2023

Experimental members: Zhang Linghao, Duan Yuxuan, Song Yinxi

① Calculation of the synthetic concentration of the probe (-trackstrand + weight)

In the standard curve (7 probes in total) of 10 nM, 2 μL of each probe is added to the 50 μL reaction system (standard curve). What is the concentration of probe required to be synthesized?

x*2/50 μL=10 nM，x=250 nM

To synthesize the 250 nM probe, how much probe needs to be added to the 60 μL synthesis system at a known concentration of 3 μM?

3 μM * x / 60 μL=0.25 μM=250 nM，x=5 μL

② Calculation of reporter probe (fluorescent FAM / ROX + BHQ)

The concentrations are as follows: 1. 10 nM，4. 40 nM，5. 50 nM

When all these are added to the standard curve system, 10 + 40 + 50 is 100 nM, and 100 nM needs to be reacted, so add 120 nM to ensure the reaction can be completed.

What is the concentration of the reporter probe (fluorescence)?

x* 2/50 = 120 nM (total concentration should be 120 nM), resulting in x = 3000 nM

With the existing fluorescent code (concentration 10 μM), how much μL to add to the 50 μL system to synthesize the probe?

10 μM x 10 ^ 3 (converted to nM) / 50 μL (system) = 3000 nM, yielding x=15 μL

1）The initial strand concentration was 3 μM in μL, and the probe weight concentration was 250 nM in μL

		Track strand	Code_F (3μM) weight molecular	Buffer	Water	Total
FAM	ARG1		5	5	6	44
FAM	CD177	5	20	6	29	60
FAM	VNN1	5	25	6	24	60
			Code_R (3μM)
ROX	IFIT1		5	5	6	44
ROX	TRDV3	5	5	6	44	60
ROX	SIGLEC1	5	20	6	29	60
ROX	LY6E	5	20	6	29	60

2）The initial strand concentration was 10 μM in μL, and the synthetic reporter probe concentration was 2.5 μM in μL

FAM	Code2_FAM （10μM）	Code2_BHQ （10μM）	Buffer	Water	Total
FAM	15	15	6	24	60
ROX	Code2_ROX （10μM）	Code2_BHQ （10μM）	Buffer	Water	Total
ROX	15	15	6	24	60

③Weight final quantitative curve, 50 μL system, unit: μL**

7 x Weight weight probe.	2 x REP (report)Fluorescent reporter probe.	Buffer	Water	dNTPs	Input	BST
7*2=14μL	2*2=34 μL	3	23 or 22	2	3 or 4	1

FAM quantification (INPUT = 3, namely target adding the 3 gene segments)

	control	1	4	5	1+4	1+5	4+5	1+4+5
order number	1.2.3.	4.5.6.	7.8.9.	10.11.12.	13.14.15	16.17.18	19.20.21	22.23.24

ROX quantification (INPUT = 4, namely target adding the 4 gene segments)

	blank	1	1+1	4	1+4	1+1+4	4+4	1+4+4	1+1+4+4
NO	1.2.3.	4.5.6.	7.8.9.	10.11.12.	13.14.15	16.17.18	19.20.21	22.23.24	25.26.27

100 bases in the conserved sequence of 100 nt seven genes (nt refers to single stranded, bp refers to duplex)

Seven genes are different, and 30 nt fragments out of 100 nt were picked out as target

Approximately linear isoscaled amplification of targets by LATE-PCR

Experimental date: 4.5.2023

Experimental members: Gui Yuyin, Huang Jiong, Ouyang Weiyi

① Dilution, in preparation for the LATE-PCR**

Primers (FP, RP) for 7 genes were supplied with ddH2O corresponding to 100 μM liquid. Then individually diluted to 10 μM. The RP was again diluted to 1 μM.(For the following LATE-PCR experiment, see the experiments in 11.4.2023)

Synthesize the weight probes. Unit : μL,

	Code（weight molecular） 5μM single strand	Track strand 5μM single strand	Buffer 10×	Water
ARG1(+1)	2	2	4	32
CD177(+4)	2x4=8	2	4	Fill complete
VNN1(+5)	2x5=10	2	4
IFIT1(-1)	2	2	4
TRDV3(-1)	2	2	4
LY6E(-4)	8	2	4
SIGLEC1(-4)	8	2	4

Synthesis Fluorescent reporter probe. Unit: μL previously used BHQ1, this time use BHQ2

Code-FAM 10μM	Code-BHQ1 10μM	Code-ROX 10μM	Code-BHQ2 10μM	Water	Buffer
15	15	0	0	15	5
0	0	15	15	15	5

weight 1. 10nM

weight 4. 40nM

weight 5. 50nM

When all these components are added to the standard curve system, 10 + 10 + 40 + 50 is 100 nM, and 100 nM needs to be reacted, so add 120 nM to ensure the reaction was complete

What concentration of the reporter probe (fluorescence) should be synthesized?

x * 2 / 50 = 120 nM (total concentration should be 120 nM), resulting in x = 3000 nM

With 10 μM fluorescent code, how much μL is needed to add to the 50μL system to synthesize the probe?

10 μM x 10 ^ 3 (converted to nM) / 50 μL (system) = 3000 nM, yielding x=15 μL

② Quantitative fluorescence curve in μL, 10 nM, x * 2 μL / 50 μL

= 10 nM for each weight probe.**

10nM per weight probe.

Genes corresponding to each target	Target (Input)	Weight probe	Fluorescent reporter probe.	Buffer 10×	Water	Enzyme 8000u/mL	dNTP （2.5mM）
A	2	7 Species x2	2Speciesx2	5	23	1	1
C	2	7 Species x2	2Speciesx2	5	23
V	2	7 Species x2	2Speciesx2	5	23
A1+C4	2+2	7 Species x2	2Speciesx2	5	21
C4+V5	2+2	7 Species x2	2Speciesx2	5	21
A1+C4+V5	2+2+2	7 Species x2	2Speciesx2	5	19
i1	2	7 Species x2	2Speciesx2	5	Fill complete
i1+T1	2+2	7 Species x2	2Speciesx2	5
S4	2	7 Species x2	2Speciesx2	5
T1+L4	2+2	7 Species x2	2Speciesx2	5
i1+T1+L4	2+2+2	7 Species x2	2Speciesx2	5	19
L6+S4	2+2	7 Species x2	2Speciesx2	5	21
T1+L6+S4	2+2+2	7 Species x2	2Speciesx2	5	19
i1+T1+L6+S4	2+2+2+2	7 Species x2	2Speciesx2	5	17

FAM（green）ROX（orange）

gain value of FAM is 6.67, gain value of ROX is 8.67 (0.33 increase except for integer change)

The synthetic probe (60μL) was synthesized at a probe concentration of 250 nM
Code（weight molecular）（5μM） Track strand （5μM） Buffer 10× Water
ARG1(+1) 3 3 6 48
CD177(+4) 3*4=12 3 6 Fill complete
VNN1(+5) 3*5=15 3 6
IFIT1(-1) 3 3 6
TRDV3(-1) 3 3 6
LY6E(-4) 12 3 6
SIGLEC1(-4) 12 3 6

	Code（weight molecular）（5μM）	Track strand （5μM）	Buffer 10×	Water
ARG1(+1)	3	3	6	48
CD177(+4)	3*4=12	3	6	Fill complete
VNN1(+5)	3*5=15	3	6
IFIT1(-1)	3	3	6
TRDV3(-1)	3	3	6
LY6E(-4)	12	3	6
SIGLEC1(-4)	12	3	6

For 10 nM in total, add 2 μL into the reaction system (standard curve), what concentration of probe is required?

x*2/50=10 nM，x=250 nM

To synthesize the 250 nM probe, how much μL needs to be added to the 60 μL system

5μM * x / 60 μL=0.25μM=250 nM，x=3 μL

Existing code (concentration 5μM), how to match 250 nM?

38 Water + 2 μL of code (concentration 5 μM) yielded 250 nM, 2 μL * 5 μM / (2 μL + 38 μL) =0.25 μM = 250 nM

Synthesize fluorescent probe (60 μL in total)
Code-FAM （10 μM） Code-BHQ1 （10 μM） Code-ROX （10 μM） Code-BHQ2 （10 μM） Water Buffer
18 18 0 0 18 6
0 0 18 18 18 6

Code-FAM （10 μM）	Code-BHQ1 （10 μM）	Code-ROX （10 μM）	Code-BHQ2 （10 μM）	Water	Buffer
18	18	0	0	18	6
0	0	18	18	18	6

2 μL added to the reaction system (standard curve), what concentration of the reporter probe (fluorescence) should be synthesized?

x*2/50=120 nM, resulting in x=3000 nM

At a concentration of 10 μM, how muchμL should be taken into the 60 μL system to synthesize the probe?

10 μM x 10 ^ 3 (converted to nM) / 60 μL (system) = 3000 nM, yielding x=18 μL

11.5.2023

Experimental date: 11.5.2023

Experimental members: Gui Yuyin, Zhou Mingzhen, Song Yinxi

① Dilution

ARG1 Template Diluted from 25 nM = 25,000 pM to 500PM, 50PM, and 5PM

Take 2μL ARG1 Template solid into 200mL EP tube, add 98μL ddH2O, mix well to obtain 500PM ARG1 Template.
10 μL 500PM ARG1 Template were taken and added to 90μL ddH2O to yield a 50PM concentration.
10 μL 50PM ARG1 Template were taken and added to 90μL ddH2O to yield a 5PM concentration.

② The ASY / LATE-PCR reaction system configuration**

TEST: ARG 1 gene, new FP and RP were selected for validation

Unit：μL，reaction system：50μL

Group 1（Into the slot C of the PCR instrument)

	FP（10μM）	RP（1μM）	Template （100nt）	Q5 high-fidelity 2X Master Mix	Water
Template concentration of 500PM	2.5	2.5	1	25	19
Template concentration of 50PM	2.5	2.5	1	25	19
Template concentration of 5PM	2.5	2.5	1	25	19
control	2.5	2.5	Add water	25	20

Group 2（Into the slot A of the PCR instrument）

	FP（10μM）	RP（1μM）	Template （100nt）	Q5 high-fidelity 2X Master Mix	Water
Template concentration of 500PM	2.5	2.5	1	25	19
Template concentration of 50PM	2.5	2.5	1	25	19
Template concentration of 5PM	2.5	2.5	1	25	19
control	2.5	2.5	Add water	25	20

③ ASY/LATE-PCR（50cycles）

A-slot setting: 2 groups (LATE)

1	2	3	4	5	6	7	8	9	10	11
98℃	98℃	55℃	72℃	GOTO2	98℃	50℃	72℃	GOTO6	72℃	4℃
30s	30s	30s	30s	X10	30s	30s	30s	X40	2min	∞

Note: GOTO2x10 means 10 cycles of 2-4, GOTO6x40 means 40 cycles of 6-8

C-slot setting: 1 group (ASY)

1	2	3	4	5	6	7
98℃	98℃	50℃	72℃	GOTO2	72℃	4℃
30s	30s	30s	30s	X50	2min	∞

Note: GOTO2x50 is 2-4 steps and 50 cycles

④ The effect of performing LATE-PCR on the ARG 1 gene was

determined using electrophoresis**

Supplement: The configuration of the 20% gel for DNA electrophoresis

5mL	PAGE	10mL
2mL（unchanged）	5x buffer TBE	10mL
3mL	ddH2O	10mL
7μL（unchanged）	TEMED	Finally, add these substances, and then the glue solidifies
55μL（unchanged）	APS	Finally, add these substances, and then the glue solidifies

Experimental configuration: 10% glue, the configuration should be shaken
2.5mL PAGE 10mL
2mL（unchanged） 5x buffer TBE 10mL
5.5mL（Fill 10mL） ddH2O 10mL
7μL（unchanged） TEMED Finally add
55μL（unchanged） APS Finally add
At the end of LATE-PCR, the above preparation can be made, and 1 μL 6x loading buffer will be added to the bottom of 9 EP tubes, and we should remember no to hang the liquid to the wall or miss it;Another 8 samples after 5 μL marker and 5 μL LATE-PCR were added to 9 EP tubes, also driving into the bottom and blowing.
Pour an appropriate amount of 0.5 x TBE (no Mg2 +) into the electrophoresis tank, add 3.5 μL sample and marker to the solidified glue (add 5 μL sample before, can not run out, so add less), set 120V voltage, running glue for 60 min.
After electrophoresis, take 30 mL of 0.5 x TBE (as in the electrophoresis tank), add to the small box, remove the glue and put it into the small box, add 3 μL of syn Golden, shake the color for 20min
After shaking, the electrophoretic gel was irradiated.

May 14th,2023

Experimental date: May 14th,2023

Experimental members: Gui yuyin, Duan Yuxuan, Song Yinxi

① Asymmetric Asym-PCR and LATE-PCR annotation**

Chart1. LATE-PCR

1	2	3	4	5	6	7	8	9	10	11
98℃	98℃	55℃	72℃	GOTO2	98℃	50℃	72℃	GOTO6	72℃	4℃
30s	30s	30s	30s	X10	30s	30s	30s	X40	2min	∞

Note: GOTO2x10,which means10 cycles of 2-4; GOTO6x40,which means 40 cycles of 6-8

Chart2. Asymmetric PCR

 1         2         3         4         5             6          7

 --------- --------- --------- --------- ------------- ---------- -------

 98℃       98℃       50℃       72℃       GOTO2         72℃        4℃

0s       30s       30s       30s       X50           2min       ∞

Note: GOTO2x50,which means 2-4 steps and 50 cycles

② Validation of the fluorescence of the asymmetric/LATE-PCR

products**

The ARG1 gene was used as a test to see whether the primer design was reasonable, that is, whether it would leak

1. Synthesize weight probes (P1) and Fluorescent reporter probe (P2)

Weight probe. (P1) Unit :μL

+-----------------+-----------------+-----------------+-----------------+
| ARG1-track      | Code（权重）    | Buffer 10x       | Water           |
| strand          |                 |                 |                 |
|                 | 5μM             |                 |                 |
| 5μM             |                 |                 |                 |
+=================+=================+=================+=================+
| 6               | 6               | 6               | 42              |
+-----------------+-----------------+-----------------+-----------------+

Fluorescent reporter probe.（P2）Fluorescent reporter probe，unit: μL

+-----------------+-----------------+-----------------+-----------------+
| FAM-code        | BHQ1-code       | Buffer 10x      | Water           |
|                 |                 |                 |                 |
| 10μM            | 10μM            |                 |                 |
+=================+=================+=================+=================+
| 3               | 3               | 6               | 48              |
+-----------------+-----------------+-----------------+-----------------+

2. Reaction system (fluorescence curve), unit: μL

           P1 final        P2 final        target   bst   dNTP   Buffer   ddH2O
           concentration   concentration                         10x
           was 20 nM       was 20 nM

---------- --------------- --------------- -------- ----- ------ -------- --------

10pM       2               2               5        1     1      5        34

1pM        2               2               5        1     1      5        34

0.1pM      2               2               5        1     1      5        34

No         2               2               5        1     1      5        34
template

Control,   2               2               0        1     1      5        39
without
target

Notice：In the primer test, we know the amount of target, and if we only test one gene (such as ARG1 this time) to observe the effect of the primer design, we do not need to add all the probes in order to save the reagent.In the process of simulating real-life diagnostic testing, we need to add all the probes.

Setting of PCR instrument when synthetic probes

+------------------+-----------+-------------+-------------+----------+
| 1                | 2         | 3           | 4           | 5        |
+==================+===========+=============+=============+==========+
| 90℃Inc -1℃/Cyc | GOTO1     | 37℃       | 25℃         | 4℃       |
+------------------+-----------+-------------+-------------+----------+
| 1min             | X53       | 5min        | 5min        | ∞        |
+------------------+-----------+-------------+-------------+----------+

Note: Inc-1℃/Cyc means minus 1℃ after the end of each cycle，that was 90(1min), 89(1min), 88(1min)......38(1min)

Concentrations corresponding to the serial number of the qPCR instrument for the fluorescence quantification reaction

	10pM asymmetric	1pM asymmetric	0.1pM asymmetric	10pM LATE	1pM LATE	0.1pM LATE	Control,without target	No template, asymmetric	no template LATE
No.	1.2.3.	4.5.6.	7.8.9.	10.11.12.	13.14.15	16.17.18	19.20.21	22.23.24	25.26.27

No.

1.2.3.

4.5.6.

7.8.9.

10.11.12.

13.14.15

16.17.18

19.20.21

22.23.24

25.26.27

④ result

AGR 1 did not leak (i. e. no fluorescence production in the absence of template), but the primer design still needs to be optimized.

June 12th, 2023

Experimental date: June 12th, 2023

Experimental members: Zhao Kexu, Huang Jiong

① synthesize the probe

+-----------+----------+----------+-----------+-----------+-----------+
| F-20      | Q-20     | V-track  | V-track   | Buffer    | Water     |
|           |          | strand1  | strand2   |           |           |
| 5μM       | 5μM      |          |           | 10x       |           |
|           |          | 5μM      | 5μM       |           |           |
+===========+==========+==========+===========+===========+===========+
| 3.5       | 3.5      | 3.5      | 3.5       | 7         | 49        |
+-----------+----------+----------+-----------+-----------+-----------+

Note: F: Q: S1: S2 is 1:1:1:1, the reason refer to the first probe structure diagram

The system has a total of 70 μl, and then it can be put into the PCR instrument, using the annealing program, about an hour of synthesis.

$C:\Users\赵可旭\AppData\Local\Temp\WeChat Files\8ad5eb3c2a37fd17c7199a7adf7c749.jpg$

1）Pre-experiments were performed on the PCR products

The pre-experiment was a step before measuring the fluorescence of all PCR products. There were only two sets of data, namely high-plus template (template is the original fragment of our PCR P), low-plus Water, which is one high and low curves. After obtaining these two curves, we can adjust the gain value in the computer. Adjusting this gain value can make our curve look better and easier to see the difference.

$C:\Users\赵可旭\AppData\Local\Temp\WeChat Files\aeef85a6e36b173bfb6e337614b414e.jpg$

2）The fluorescence of the PCR products was measured

In total, we measured 30 groups of fluorescence, namely 4 groups of late and asymmetry, respectively measured 3 large groups of parallel control, at the same time, the positive control group, namely template as target, and the blank control group and Water , each do 3 groups of parallel. So in total, 4X3 + 4X3 + 1X3 + 1X3=30 groups。

Target            Probe             Buffer            Water
----------------- ----------------- ----------------- -----------------
2                 2                 5                 42

② result

The fluorescence increased during pre-experiment; however, neither the positive positive control and the samples after PCR. Therefore, further optimization of the experimental design is required

Weight subtraction

Experimental date: June 17th,2023

Experimental members: Zhang Linghao, Duan Yuxuan, Song Yinxi, Zhang Keyue

Total of 50 μL system

  7 × W           2 × REP        Buffer    Water     dNTPs     input    BST
  --------------- -------------- --------- ------- --------- -------- ------
  7×2=14 μL       2×2=4 μL       3         16      2         10       1

The initial strand concentration was 5 μM, and the synthetic weight probe concentration was 250 nM

                      Track       F_code2      Buffer    Water      total
                      strand
-------- ------------ ----------- ------------ --------- -------- ---------
FAM      ARG1         2           2            4         32       40 μL

         CD177        2           8            4         26       40 μL

         VNN1         2           10           4         24       40 μL

                      Track       R_code2      Buffer    Water      total
                      strand

ROX      LY6E         2           8            4         26       40 μL
2
         SIGLEC1      2           8            4         26       40 μL

         IFIT1        2           2            4         32       40 μL

         TRDV3        2           2            4         32       40 μL

The initial strand concentration was 10 μM, and the synthetic reporter probe concentration was 2.5 μM

  --------------------------------------------------------------------------

  FAM      code2_FAM        code2_BHQ         Buffer     Water      total

  -------- ---------------- ----------------- ---------- -------- ----------

           10               10                4          16       40 μL

  ROX      code2_R          code2_Q2          Buffer     Water      total

           10               10                4          16       40 μL

  --------------------------------------------------------------------------

Mixed Late-PCR

 -----------------------------------------------------------------------
 BI 1                            Q5 2× Mix                       25
 ------------------------------- ------------------------------- -------
 A10 C10 V10 L1 S1 I7 T3         25 μM A-F                       1
 BI 2                            2.5 μM A-R                      1
 A2 C5 V7 L1 S1 I3 T1            C-F                             1
 VI 1                            C-R                             1
 A3 C2 V1 L10 S10 I5 T5          V-F                             1
 VI 2                            V-R                             1
 A3 C1 V1 L5 S8 I3 T6            L-F                             1
 N 1                             L-R                             1
 A1 C1 V1 L1 S1 I1 T1            S-F                             1
 N 2                             S-R                             1
 A1 C2 V2 L2 S2 I2 T1            I-F                             1
                                 I-R                             1
                                 T-F                             1
                                 T-R                             1
                                 500 pM Template (7 Genes)       7
                                 Water                           4

Engineering Cycle 2: A molecular classifier used for lung adenocarcinoma detection

DNA electrophoresis

of hsa-mir-192-5p、hsa-mir-375-3p、hsa-mir-21-5p、hsa-let-7a-2-5p、hsa-let-7b-5p、hsa-mir-143-3p

Experimental date: Aug.23th 2023

Experimental members: Ouyang Weiyi, Chu Zibin, Zhang Keyue, Wang Yinhao

① Configured gel solution (15% glue)

    -----------------------------------------------------------------------

    4.25ml               ddH2O

3.75ml 40%Acrylamide (gel 10mL solution)

    2ml                  5×TBE

                                                    -----------------------

    7μL                  TEMED                      Finally add it, after
                                                    adding it, the glue
                                                    stared to solidify

  55μL                 10%APS
  -----------------------------------------------------------------------

Ps: Remember to insert a 10-well electrophoresis comb after pouring in the glue

③ Configure sample (while waiting for gel in 15 minutes)

-----------------------------------------------------------------------

1μL loadingbuffer                   4  μL of the mixture of
                                    loadingbuffer and sample was added
                                    to the wells

-----------------------------------

5μL sample                          4  μL of the mixture of
                                    loadingbuffer and sample was added
                                    to the wells

-----------------------------------------------------------------------

A total of three glue were run, and samples in each glue were as follows

(1)：

--------------------------------------------------------------------------

maker          F-code2        192-5p-track   P1(192-5p)     21-5-track
                              strand                        strand

-------------- -------------- -------------- -------------- --------------

P1(21-5p)      375-3p-track   P1(375-3p)
               strand

--------------------------------------------------------------------------

(2)：

--------------------------------------------------------------------------

maker          H-coder        7a2-code1      7a2-5p-track   7a2 bloker
                                             strand

-------------- -------------- -------------- -------------- --------------

7a2 PROBE2     P1(7a2)        P2(7a2)

--------------------------------------------------------------------------

(3)：

--------------------------------------------------------------------------

maker          H-coder        7b-5p-code1    7b-5p-track    7b-5p bloker
                                             strand

-------------- -------------- -------------- -------------- --------------

7b-5p PROBE2   P1 7b-5p       P2 7b-5p       143-3p-track   P2(143-3p)
                                             strand         probe

--------------------------------------------------------------------------

1.Pour 0.5×TBE into the electrophoresis tank, add 4 μL of the configured sample, and set the 120V voltage for 80min.

2.After electrophoresis, take 30 mL of 0.5xTBE (as in the electrophoresis tank), add to the small box, add 3 μL of syn Golden dye, shake the glue into it, and shake the color with tin foil for 20min.

3.After shaking, the electrophoretic gel was irradiated.

results：

8.23 楠岃瘉鎺㈤拡缁撴瀯-1 80min 15%
1

Probe synthesis, and fluorescence curve determination

Experimental date: Aug.24th,2023

Experimental members: Ouyang Weiyi, Chu zibin, Wang Yinhao, Hu Haoyang, Liu Chenyuan

① Synthesize the probe

All samples were 10 μM (already diluted)

Calculation: The sample volume was 60 μL×250 nM = 10 μM , in order to get slightly more fluorescence, we used about 0.5 μL.

1.hsa-mir-192-5p(+1)

+----------+-----------+-----------+-----------+----------+-----------+
| P1       | 192-track | F-code2   | Buffer    | Water    | 60μL      |
|          | strand    |           |           |          |           |
|          |           |           |           |          | 250nM     |
|          |           |           |           |          |           |
|          |           |           |           |          | System    |
+==========+===========+===========+===========+==========+===========+
|          | 1.5μL     | 1.5μL     | 6μL       | 51μL     |           |
+----------+-----------+-----------+-----------+----------+-----------+
| P2       | FAM       | BHQ       | Buffer    | Water    |           |
+----------+-----------+-----------+-----------+----------+-----------+
|          | 2μL       | 2μL       | 6μL       | 50μL     |           |
+----------+-----------+-----------+-----------+----------+-----------+

2.hsa-let-7a-2-5p(-5×-5)

+----+-------------+---------------+--------+--------+-----+---------+
| P1 | 7a-track    | 7a-CODE-1     | Buffer | Water  |     | 60μL    |
|    | strand      |               |        |        |     |         |
|    |             |               |        |        |     | System  |
+====+=============+===============+========+========+=====+=========+
|    | 1.5μL       | 7.5μL         | 6μL    | 45μL   |     |         |
+----+-------------+---------------+--------+--------+-----+---------+
| P2 | bloker      | H-code2       | Probe2 | Buffer | H2O | 60μL    |
|    |             |               |        |        |     | 1250nM  |
|    |             |               |        |        |     | System  |
+----+-------------+---------------+--------+--------+-----+---------+
|    | 7.5μL       | 37.5μL        | 7.5μL  | 6μL    | 1.  |         |
|    |             |               |        |        | 5μL |         |
+----+-------------+---------------+--------+--------+-----+---------+
| P3 | HEX(100μM)  | BHQ2(100μM)   | Buffer | Water  |     | 60μL    |
|    |             |               |        |        |     |         |
|    |             |               |        |        |     | 6250nM  |
|    |             |               |        |        |     |         |
|    |             |               |        |        |     | System  |
+----+-------------+---------------+--------+--------+-----+---------+
|    | 4μL         | 4μL           | 6μL    | 46μL   |     |         |
+----+-------------+---------------+--------+--------+-----+---------+
| P4 | H-code2     | Probe2        | Buffer | Water  |     | 60μL    |
|    |             |               |        |        |     |         |
|    |             |               |        |        |     | 1250nM  |
|    |             |               |        |        |     |         |
|    |             |               |        |        |     | System  |
+----+-------------+---------------+--------+--------+-----+---------+
|    | 37.5μL      | 7.5μL         | 6μL    | 1.5μL  |     |         |
+----+-------------+---------------+--------+--------+-----+---------+

3.hsa-mir-21-5p(+3)

+-------+--------------+------------+---------+----------+-----------+
| P1    | 21-track     | F-code2    | Buffer  | Water    | 60μL      |
|       | strand       |            |         |          |           |
|       |              |            |         |          | 250nM     |
|       |              |            |         |          |           |
|       |              |            |         |          | System    |
+=======+==============+============+=========+==========+===========+
|       | 1.5μL        | 4.5μL      | 6μL     | 48μL     |           |
+-------+--------------+------------+---------+----------+-----------+
| P2    | FAM          | BHQ        | Buffer  | Water    | 60μL      |
|       |              |            |         |          |           |
|       |              |            |         |          | 750nM     |
|       |              |            |         |          |           |
|       |              |            |         |          | System    |
+-------+--------------+------------+---------+----------+-----------+
|       | 5μL          | 5μL        | 6μL     | 44μL     |           |
+-------+--------------+------------+---------+----------+-----------+

4.hsa-mir-375-3p(+2)

+-------+--------------+------------+---------+-----------+-----------+
| P1    | 375-track    | F-code2    | Buffer  | Water     | 60μL      |
|       | strand       |            |         |           |           |
|       |              |            |         |           | 250nM     |
|       |              |            |         |           |           |
|       |              |            |         |           | System    |
+=======+==============+============+=========+===========+===========+
|       | 1.5μL        | 3μL        | 6μL     | 49.5μL    |           |
+-------+--------------+------------+---------+-----------+-----------+
| P2    | FAM          | BHQ        | Buffer  | Water     | 60μL      |
|       |              |            |         |           |           |
|       |              |            |         |           | 500nM     |
|       |              |            |         |           |           |
|       |              |            |         |           | System    |
+-------+--------------+------------+---------+-----------+-----------+
|       | 3.5μL        | 3.5μL      | 6μL     | 47μL      |           |
+-------+--------------+------------+---------+-----------+-----------+

5.hsa-mir-143-3p(-3)

+-------+--------------+------------+---------+----------+-----------+
| P1    | 143-track    | H-code2    | Buffer  | Water    | 60μL      |
|       | strand       |            |         |          |           |
|       |              |            |         |          | 250nM     |
|       |              |            |         |          |           |
|       |              |            |         |          | System    |
+=======+==============+============+=========+==========+===========+
|       | 1.5μL        | 4.5μL      | 6μL     | 48μL     |           |
+-------+--------------+------------+---------+----------+-----------+
| P2    | HEX          | H-BHQ      | Buffer  | Water    | 60μL      |
|       |              |            |         |          |           |
|       |              |            |         |          | 750nM     |
|       |              |            |         |          |           |
|       |              |            |         |          | System    |
+-------+--------------+------------+---------+----------+-----------+
|       | 5μL          | 5μL        | 6μL     | 44μL     |           |
+-------+--------------+------------+---------+----------+-----------+

6.hsa-let-7b-5p(-4×-2)

+----+-------------+---------------+--------+--------+-----+---------+
| P1 | 7b-track    | 7b-CODE-1     | Buffer | Water  |     | 60μL    |
|    | strand      |               |        |        |     |         |
|    |             |               |        |        |     | 250nM   |
|    |             |               |        |        |     |         |
|    |             |               |        |        |     | System  |
+====+=============+===============+========+========+=====+=========+
|    | 1.5μL       | 6μL           | 6μL    | 46.5μL |     |         |
+----+-------------+---------------+--------+--------+-----+---------+
| P2 | bloker      | H-code2       | Probe2 | Buffer | H~  | 60μL    |
|    |             |               |        |        | 2~O |         |
|    |             |               |        |        |     | 1000nM  |
|    |             |               |        |        |     |         |
|    |             |               |        |        |     | System  |
+----+-------------+---------------+--------+--------+-----+---------+
|    | 6μL         | 12μL          | 6μL    | 6μL    | 3   |         |
|    |             |               |        |        | 0μL |         |
+----+-------------+---------------+--------+--------+-----+---------+
| P3 | HEX         | BHQ2          | Buffer | Water  |     | 60μL    |
|    |             |               |        |        |     |         |
|    |             |               |        |        |     | 2000nM  |
|    |             |               |        |        |     |         |
|    |             |               |        |        |     | System  |
+----+-------------+---------------+--------+--------+-----+---------+
|    | 12.5μL      | 12.5μL        | 6μL    | 29μL   |     |         |
+----+-------------+---------------+--------+--------+-----+---------+
| P4 | H-code2     | Probe2        | Buffer | Water  |     | 60μL    |
|    |             |               |        |        |     |         |
|    |             |               |        |        |     | 1000nM  |
|    |             |               |        |        |     |         |
|    |             |               |        |        |     | System  |
+----+-------------+---------------+--------+--------+-----+---------+
|    | 12μL        | 6μL           | 6μL    | 36μL   |     |         |
+----+-------------+---------------+--------+--------+-----+---------+

After the configuration is completed, put them into the PCR instrument, select YHX-90-1.td, the reagent capacity of 60 μL, and start running.

② Fluorescence curve determination**

Three parallel experiments were performed for each gene, two cascades with both bloker and no bloker, and three sets of blank controls, a total of 27 groups. The targets were all already diluted 10 nM.

21、375、143：

--------------------------------------------------------------------------

Target   P1       P2       dNTPs    10×Buffer   Enzyme   Water      50μL

-------- -------- -------- -------- ----------- -------- -------- --------

1μL      2μL      2μL      2μL      5μL         1μL      37μL       50μL

--------------------------------------------------------------------------

7a1、7b1(added bloker)：

| Target   | P1   | P2   | P3   | dNTPs ×Buffer | 10      | Enzyme    | Water  | 50 μL  |
+======+======+======+======+=======+=========+=======+======+=====+
| 1μL  | 2μL  | 2μL  | 2μL  | 2μL   | 5μL     | 1μL   | 35μL |     |
+------+------+------+------+-------+---------+-------+------+-----+

7a2、7b2(nobloker)：

+------+------+------+------+-------+---------+-------+------+-----+
| Ta   | P1   | P3   | P4   | dNTPs | 10      | E     | Water  | 50  |
| rget |      |      |      |       | ×Buffer | nzyme |      |     |
|      |      |      |      |       |         |       |      | μL  |
+======+======+======+======+=======+=========+=======+======+=====+
| 1μL  | 2μL  | 2μL  | 2μL  | 2μL   | 5μL     | 1μL   | 35μL |     |
+------+------+------+------+-------+---------+-------+------+-----+

blank：

 --------------------------------------------------------------------------

 Any P1     Any P2      dNTPs    10×Buffer     Enzyme     Water    50μL

 ---------- ----------- -------- ------------- ---------- ------ ----------

 2μL        2μL         2μL      5μL           1μL        38μL    50μL

 --------------------------------------------------------------------------

The enzyme should be added to the wall of the tube, and then put into qPCR instrument after centrifugation.

The qPCR instrument was adjusted in advance, with cycles of 1000,25 degrees and 5s per cycle. Green fluorescence gain 7.67 and yellow fluorescence gain 9.33.

	Fluorescent reporter probe 600nM	weight molecular 500nM	Buffer 10x	ddH2O
0	10	0	5	35
10	10	1	5	34
20	10	2	5	33
30	10	3	5	32
40	10	4	5	31
50	10	5	5	30
60	10	6	5	29
70	10	7	5	28
80	10	8	5	27
90	10	9	5	26
100	10	10	5	25

	Fluorescent reporter probe 600nM	weight molecular 500nM	Buffer 10x	ddH2O
0	10	0	5	35
10	10	1	5	34
20	10	2	5	33
30	10	3	5	32
40	10	4	5	31
50	10	5	5	30
60	10	6	5	29
70	10	7	5	28
80	10	8	5	27
90	10	9	5	26
100	10	10	5	25

Engineering Cycle 1: A molecular classifier used to distinguish between bacterial or viral infections​

Electrophoresis verification of the weight probe construction​

Probe synthesis​

Electrophoresis verification​

① Configure gel solution (15% electrophoresis gel)​

② Configure sample (while waiting for gel in 15 minutes)​

Report Probes in response to weight molecular via the TMSD reaction​

Synthesis the probe​

Reaction system preparation​

Detection and weighting of the target signal by weight probes​

Synthetic weight probes. (P1) and the fluorescent reporter probe. (P2)​

Reaction system preparation​

Fluorescent validation of the weight addition​

① Calculation of the synthetic concentration of the probe (-trackstrand + weight)​

② Calculation of reporter probe (fluorescent FAM / ROX + BHQ)​

③Weight final quantitative curve, 50 μL system, unit: μL**​

Approximately linear isoscaled amplification of targets by LATE-PCR​

① Dilution, in preparation for the LATE-PCR**​

② Quantitative fluorescence curve in μL, 10 nM, x * 2 μL / 50 μL​

11.5.2023​

① Dilution​

② The ASY / LATE-PCR reaction system configuration**​

③ ASY/LATE-PCR（50cycles）​

④ The effect of performing LATE-PCR on the ARG 1 gene was​

May 14th,2023​

① Asymmetric Asym-PCR and LATE-PCR annotation**​

② Validation of the fluorescence of the asymmetric/LATE-PCR​

④ result​

June 12th, 2023​

① synthesize the probe​

② result​

Weight subtraction​

Engineering Cycle 2: A molecular classifier used for lung adenocarcinoma detection​

DNA electrophoresis​

① Configured gel solution (15% glue)​

Probe synthesis, and fluorescence curve determination​

① Synthesize the probe​

② Fluorescence curve determination**​

Engineering Cycle 1: A molecular classifier used to distinguish between bacterial or viral infections

Electrophoresis verification of the weight probe construction

Probe synthesis

Electrophoresis verification

① Configure gel solution (15% electrophoresis gel)

② Configure sample (while waiting for gel in 15 minutes)

Report Probes in response to weight molecular via the TMSD reaction

Synthesis the probe

Reaction system preparation

Detection and weighting of the target signal by weight probes

Synthetic weight probes. (P1) and the fluorescent reporter probe. (P2)

Reaction system preparation

Fluorescent validation of the weight addition

① Calculation of the synthetic concentration of the probe (-trackstrand + weight)

② Calculation of reporter probe (fluorescent FAM / ROX + BHQ)

③Weight final quantitative curve, 50 μL system, unit: μL**

Approximately linear isoscaled amplification of targets by LATE-PCR

① Dilution, in preparation for the LATE-PCR**

② Quantitative fluorescence curve in μL, 10 nM, x * 2 μL / 50 μL

11.5.2023

① Dilution

② The ASY / LATE-PCR reaction system configuration**

③ ASY/LATE-PCR（50cycles）

④ The effect of performing LATE-PCR on the ARG 1 gene was

May 14th,2023

① Asymmetric Asym-PCR and LATE-PCR annotation**

② Validation of the fluorescence of the asymmetric/LATE-PCR

④ result

June 12th, 2023

① synthesize the probe

② result

Weight subtraction

Engineering Cycle 2: A molecular classifier used for lung adenocarcinoma detection

DNA electrophoresis

① Configured gel solution (15% glue)

Probe synthesis, and fluorescence curve determination

① Synthesize the probe

② Fluorescence curve determination**

	Fluorescent reporter probe 600nM	weight molecular 500nM	Buffer 10x	ddH2O
0	10	0	5	35
10	10	1	5	34
20	10	2	5	33
30	10	3	5	32
40	10	4	5	31
50	10	5	5	30
60	10	6	5	29
70	10	7	5	28
80	10	8	5	27
90	10	9	5	26
100	10	10	5	25