Notebook

11^th

- Culture E. coli

12^th

- Cas12a protein expression:

• Addition of IPTG
• Test OD₆₀₀ value of pET-28a integrated E. coli BL21(DE3)

- pUC57 plasmid extraction:

• Measure the concentration using Nanodrop

PCR of cagA, ipaH, invA, 16S_ pUC57 plasmids

13^th

- Cas12a protein extraction:

• Ultrasound lysis
• Nickel affinity purification

- Gel electrophoresis of cagA, ipaH, invA, and 16S plasmids:

• Extraction of target DNA fragments
• Measure the concentration using Nanodrop

14^th

- Cas12a protein electrophoresis,SDS-PAGE

- cagA, ipaH, invA, and 16S DNA in vitro transcription

- sgRNA purification

15^th

- Incubation of sgRNA and Cas12a proteins

- Test the reaction system using (cagA, ipaH, invA, and 16S) pUC57 plasmid

• ssDNA was included in the system and the result will be based on fluorescent intensity

- Test the reaction system using oligo DNA (cagA, ipaH, invA, and 16S)

• verify using gel electrophoresis after PCR

16^th

- Incubation of sgRNA and Cas12a proteins

- Test reaction system using oligo DNA (cagA, ipaH, invA, and 16S)

• prove using gel electrophoresis after PCR

- Test reaction system using (cagA, ipaH, invA, and 16S) pUC57 plasmid

• ssDNA was included in the system and the result will be based on fluorescent intensity

17^th

- Test resulting system (cagA, ipaH, invA, and 16S) pUC57 plasmid

• ssDNA was included in the system and the result will be based on fluorescent intensity detected by multiscan ascent

- Test the reaction system using bacteria culture

• Bacteria lysis
• Test PCR results using gel electrophoresis
• Add ssDNA into the system and test using multiscan ascent