TIANprep Mini Plasmid Kit Protocol
TIANGEN Biotech
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Abstract
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.
Steps
Preparation of Bacterial Cells
Centrifiguation of 1-5mL of bacterial cells in a microcentrifuge tube at 12000rpm
Direct drainage of supernatant by opening and inverting the tube.
Complete resuspension of the bacteria pellet in 250µL of Buffer P1 .
Add 250µL of Buffer P2 (lysis buffer) and mix by inverting the tube 6-8 times.
Add 350µL of Buffer P3 and mix immediately by inverting the tube 6-8 times to neutralize Buffer P2.
Centrifuge at 12000rpm
Preparation of Spin Column CP3
Place Spin Column CP3 in a clean collection tube.
Add 500µL of Buffer BL to CP3.
Centrifuge at 12000rpm
Discard the flow-through and put the Spin Column CP3 back into the collection tube.
Plasmid Extraction
Transfer of supernatant from Step 6 to Spin Column CP3 with collection tube attached.
Centrifuge at 12000rpm
Discard the flow-through, retain Spin Column CP3 with collection tube attached.
OPTIONAL: Wash the Spin Column CP3 by adding 500µL of Buffer PD and centrifuge at 12000rpm and discard the flow-through.
Wash the column with Buffer PW
Add 600µL of Buffer PW
Centrifuge at 12000rpm .=
Discard the flow-through, retain Spin Column CP3 with collection tube attached.
Repeat Step 15.
Centrifuge at 12000rpm to remove residual Buffer PW.
Air dry the column at room temperature for a while to allow residual ethanol from Buffer PW to evaporate.
Place Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. Discard the collection tube.
Add 50-100µL of Buffer EB to the center of the Spin Column CP3.
Incubate for 0h 2m 0s
Centrifuge at 12000rpm
