Seven days

Cultivation of BL21 bacteria

Eight days.

- Cas12a protein expression:

Increased IPTG

Testing the value of OD 600pET-28a synthesised E. coli BL21 (DE3)

Recombinant plasmid pET-Cas12a plasmid extraction:

Concentrations were measured using Nanodrop

PCR detection of pET-Cas12a plasmid

Nine Days.

Cas12a protein extraction:

Ultrasonic Cleavage

Nickel affinity purification

Extraction of target DNA fragments

Concentrations were measured using Nanodrop

Ten days

Cas12a protein electrophoresis, SDS-PAGE

In vitro transcription of pET-Cas12a

Purification of crRNA

Incubation of crRNA and Cas12a protein

- Test reaction system using pET-Cas12a plasmid

reporter is included in the system, results will be based on colour intensity

Reaction system tested using different miRNAs (miR-21 miR-16 miR-155 miR-221)

Validation using gel electrophoresis after PCR

Eleven days.

- Incubation of crRNA and Cas12a protein

- Test reaction system using different miRNAs (miR-21 miR-16 miR-155 miR-221)

PCR followed by gel electrophoresis proved

- Test reaction system using pET-Cas12a plasmid

reporter is included in the system, results will be based on colour intensity

Twelve days

- Test results system pET-Cas12a plasmid

reporter is included in the system, the results will be based on the intensity of colour detected by multiple scanning rises

Lateral chromatography test strip results.A. Negative. B. Positive. Negative: the T-line does not show any colour,

which is classified as negative, indicating that the reporter molecule has not been cleaved by Casase and Casase

has not been activated; Positive: the T-line shows colour, which is classified as positive,indicating that the

nucleic acid probe has been cleaved by Casase and Casase has been activated;

Notebook entry：