1.Plasmid Clone
(1) Heat shock transformation of Escherichia coli
⚫ Melt the competent Escherichia coli on the ice.
⚫ Blend the plasmid with the competent Escherichia coli,then take an ice bath for 30 minutes.
⚫ 42°C Heat for 90 s,then take an ice bath for 5 minutes.
⚫ Add 500μl nonresistant LB and put it into a 37°C rocker for 40 minutes.
⚫ Centrifuge under 7000 rpm for 1 minute.
⚫ Inhale the upper clearance and leave 40 μl heavy suspension.
⚫ Coating the board with AMP.
(2) mini shaking of the plasmid
Select a single coenobium, lift it with the tip,and put it into mixture mixed by 2 ml LB and 2μl AMP,then cultivate the cells by a 37°C 180 rpm rocker for 10 hours.
(3) large shaking of the plasmid
Cultivate the mixture mixed by 500 ml LB，500 μl AMP and 1ml shaked plasmid into a 37°C 180 rpm rocker for 16 hours.
(4) plasmid mini extraction
⚫ Centrifuge under 8000 rpm for 5 minute,then collect the cells.
⚫ Remove the supernatant and add the 250 μl Buffer P1 / RNase A mixture.
⚫ Add 250 μl Buffer P2,invert to mix and place at ordinary temperature.
⚫ Add 250 μl Buffer P2,invert to mix.
⚫ Centrifuge under 13000 g for 10 minute.
⚫ Place the HiPure DNA Mini ColumnⅡinto a collection tube,then transfer the supernatant to a column and centrifuge under 13000 g for 1 minute.
⚫ Discard the filtrate,place the column into the collection tube and add 500 μl Buffer PW1,then centrifuge under 13000 g for 1 minute.
⚫ Discard the filtrate,place the column into the collection tube and add 600 μl Buffer PW2,then centrifuge under 13000 g for 1 minute.
⚫ Discard the filtrate,place the column into the collection tube and add 300 μl Buffer PW2,then centrifuge under 13000 g for 1 minute.
⚫ Discard the filtrate,place the column into the collection tube and centrifuge under 13000 g for 1 minute.
⚫ Place the column into the collection tube and add 50 μl DEPC water,then centrifuge under 13000 g for 3 minute.
⚫ Collect the filtrate.
(5) plasmid large extraction
⚫ Add 300 ml shaked bacterial liquid into 500 ml tube.
⚫ Centrifuge under 4000 rpm for 15 minute.
⚫ Add 10 ml SolutionⅡ , mix and place at ordinary temperature for 2 min.
⚫ Add 5 ml pre-cooled N3 Buffer,mix and place at ordinary temperature for 2 min.
⚫ Discard the precipitation,then transfer the filtrate to a new 50 ml tube.
⚫ Add 0.1 x volume of ETR Solution.
⚫ Place on the ice 10 min.
⚫ 42℃ water bath for 5 min.
⚫ Centrifuge at 8000 g for 5 min at 25℃.
⚫ Transfer the supernatant to a new 50 ml tube with 0.5 x volume of absolute ethanol,mix and place at ordinary temperature for 2 min.
⚫ Place the DNA Maxi Column into a 50 m l tube,add 3 ml GPS Buffer and place at ordinary temperature for 2 min,then centrifuge at 4000 g for 3 min .
⚫ Discard the filtrate ,add 20 ml of the above-added absolute ethanol solution, and centrifuge at 5,000 g for 3 min.
⚫ Readd the filtrate to DNA Maxi Column and centrifuge at 5,000 g for 3 min.
⚫ Repeat the upper two-step process..
⚫ Add 10 ml HBC Buffer and centrifuge at 5000 g for 3 min.
⚫ Discard the filtrate,add 15 ml DNA Wash Buffer and centrifuge at 5000 g for 3 min.
⚫ Discard the filtrate,add 10 ml DNA Wash Buffer and centrifuge at 5000 g for 3 min.
⚫ Discard the filtrate,then centrifug at 5,000 g for 10 min.
⚫ Remove the DNA Maxi Column and air for 5 min.
⚫ Transfer the DNA Maxi Column to a new 50 ml tube,add 500 ml of sterile water to DNA Maxi Column and place at ordinary temperature for 2 min,then centrifuge at 10,000 g for 10 min,collect the filtrate.
⚫ Repeat the previous step three times.
2.PCR Amplification
(1) PCR system
| Agentia |
Dosage/μl |
| Taq mix |
5 |
| DEPC water |
2 |
| DNA |
1 |
| F Primer (10 μM) |
1 |
| R Primer (10 μM) |
1 |
| Total |
10 |
(2) PCR process
| 95 ℃ |
5 min |
| 95 ℃ |
30 sec |
| 58 ℃ |
30 sec |
| 72 ℃ |
15 sec |
| 72 ℃ |
5 min |
| 10 ℃ |
∞ |
3.RPA Amplification
(1) RPA system
| Agentia |
Dosage/μl |
| Buffer A |
25 |
| DEPC water |
13 |
| mix and divide into five group |
| F Primer (10 μM) |
0.4 |
| R Primer (10 μM) |
0.4 |
| DNA |
1 |
| BufferB((dilute twice and add to the tube cap) |
1 |
| 39℃ 30 min |
(2) purify the RPA product
RPA product: trichloromethane: phenol =2:1:1
Refer to the above system to add 15 μl RPA product, 7.5 μ l trichloromethane and 7.5 μ l phenol,then mix and centrifuge at 13,000 rpm for 10 min.
(3) recycle the RPA gel
⚫ Add sample into a 1.5 ml EP tube and use water to replenish to 100 μl .
⚫ Add Equal volume of Buffer GDP.
⚫ Add 1.5 x the above volume of absolute ethanol .
⚫ Place the column into the collection tube,add the mixture to the center of the column and centrifuge at 10,000 g for 60s.
⚫ Repeat the last step.
⚫ Discard the filtrate,set back the column to the tube and add 500 μl Buffer DW2,then centrifuge at 8000 g for 60s
⚫ Discard the filtrate,set back the column to the tube and add 25μl Elution Buffer and place for 2 min,then centrifuge at 10,000 g for 60s.
4.CRISPR Cas12a detection
(1) CRISPR Cas12a system for fluorescence detection
| Agentia |
Dosage/μl |
| Buffer R2.1(10×） |
0.6 |
| DEPC water |
3.6 |
| Cas protein (10 μM) |
0.3 |
| sgRNA (250 nM) |
1.5 |
| 37 ℃ 15 min |
| 15 nt probe (10 μM) |
4 |
| add 9μl after mixing |
| substrate |
1 |
| Buffer R2.1(1×） |
40 |
| 37 ℃ 30 min |
(2) CRISPR Cas12a system for test paper detection
| Agentia |
Dosage/μl |
| Buffer R2.1(10×） |
1.2 |
| DEPC water |
7.2 |
| Cas protein (10 μM) |
0.6 |
| sgRNA (250 nM) |
3.0 |
| 37 ℃ 15 min |
| 15 nt probe (10 μM) |
3.0 |
| add 9μl after mixing |
| substrate |
1 |
| Buffer R2.1(1×） |
40 |
| 37 ℃ 30 min |
In the process of using the test paper, we found that there may be a slow binding speed of the probe and colloidal gold, resulting in false positives.
So, we cut out the part of the test paper containing colloidal gold and elute the colloidal gold (50 μL DEPC water was used for each test paper).
40 μL colloidal gold solution and 40 μL digested products are mixed, and detected in 96-well plate.
C band appears within 2 minutes, and the result interpretation should be completed within 10 minutes.
5.DNaseⅠsystem
| Agentia |
Dosage/μl |
| 15 nt probe |
1 |
| 10× Buffer(include MgCl2) |
5 |
| DNaseⅠ |
2 |
| DEPC water |
add to 50 |
| 37 ℃ 15 min |
| EDTA（50mM） |
1 |
| 65℃ 10 min |
