TITLE: TIANprep Mini Plasmid Kit Protocol
AUTHORS: TIANGEN Biotech

[DESCRIPTION]
TIANprep Mini Plasmid Kit is based on alkaline lysis technology followed by adsorption of DNA onto silica membrane in the presence of high salt. Plasmid DNA purified with TIANprep Mini Plasmid Kit is immediately readyfor use. Phenol extraction and ethanol precipitation are not required. High quality plasmid DNA is eluted in a small volume of Tris Buffer or deionized water. This protocol is designed for purification of plasmid DNA from 1-5ml overnight cultures of E. coli in LB (Luria-Bertani) medium. Plasmid DNA prepared by TIANprep Mini Plasmid Kit is suitable for avariety of routine applications including restriction enzyme digestion, sequencing, library screening, ligation and trans-formation, in vitro translation, and transfection of robust cells.

[BEFORE_START]
Buffer P1 must be activated with RNase A solution before use (one vial per one bottle) and store at 2-8 degrees celsius after activation.


[GUIDELINES]
Applicable to TIANprep Mini Plasmid Kit manufactured by TIANGEN.

[STEPS]
SECTION: Preparation of Bacterial Cells
1. Centrifiguation of 1-5 mL of bacterial cells in a microcentrifuge tube at   12000 rpm, 1 min  
 

SECTION: Preparation of Bacterial Cells
2. Direct drainage of supernatant by opening and inverting the tube.
 

SECTION: Preparation of Bacterial Cells
3. Complete resuspension of the bacteria pellet in 250 µL of Buffer P1.
 

SECTION: Preparation of Bacterial Cells
4. Add 250 µL of Buffer P2 (lysis buffer) and mix by inverting the tube 6-8 times.
 

 

SECTION: Preparation of Bacterial Cells
5. Add 350 µL of Buffer P3 and mix immediately by inverting the tube 6-8 times to neutralize Buffer P2.
 

 

SECTION: Preparation of Bacterial Cells
6. Centrifuge at 12000 rpm, 10 min 
SECTION: Preparation of Spin Column CP3
7. Place Spin Column CP3 in a clean collection tube.
SECTION: Preparation of Spin Column CP3
8. Add 500 µL of Buffer BL to CP3.

SECTION: Preparation of Spin Column CP3
9. Centrifuge at 12000 rpm, 1 min 

SECTION: Preparation of Spin Column CP3
10. Discard the flow-through and put the Spin Column CP3 back into the collection tube.
SECTION: Plasmid Extraction
11. Transfer of supernatant from Step 6 to Spin Column CP3 with collection tube attached.

SECTION: Plasmid Extraction
12. Centrifuge at 12000 rpm, 1 min 

SECTION: Plasmid Extraction
13. Discard the flow-through, retain Spin Column CP3 with collection tube attached.
SECTION: Plasmid Extraction
14. OPTIONAL: Wash the Spin Column CP3 by adding 500 µL of Buffer PD and centrifuge at 12000 rpm, 1 min and discard the flow-through.

 

SECTION: Plasmid Extraction
15. Wash the column with Buffer PW
SECTION: Plasmid Extraction
15.1. Add 600 µL of Buffer PW
 

SECTION: Plasmid Extraction
15.2. Centrifuge at 12000 rpm, 1 min .=

SECTION: Plasmid Extraction
15.3. Discard the flow-through, retain Spin Column CP3 with collection tube attached.
SECTION: Plasmid Extraction
16. Repeat Step 15.

SECTION: Plasmid Extraction
17. Centrifuge at 12000 rpm, 2 min to remove residual Buffer PW.

SECTION: Plasmid Extraction
18. Air dry the column at room temperature for a while to allow residual ethanol from Buffer PW to evaporate.
 

SECTION: Plasmid Extraction
19. Place Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. Discard the collection tube.
SECTION: Plasmid Extraction
20. Add 50-100 µL  of Buffer EB to the center of the Spin Column CP3.

SECTION: Plasmid Extraction
21. Incubate for 2 min 

SECTION: Plasmid Extraction
22. Centrifuge at 12000 rpm, 2 min