Accuprep Plasmid Mini Extraction kit (K-3030, K-3030-1,bioneer)
https://www.bioneer.co.kr/literatures/manual/AccuPrep/UG_AccuPrep_Plasmid_Mini_Extraction_Kit.pdf
PA1 buffer (Resuspension), RNaseA 포함 후 4°C
P2 buffer (Lysis)
PA3 buffer (Neutralizion)
W2 buffer (Washing)
EA buffer (Elution), 사용 전 Pre-heat 60°C
1. Harvest the 1-5ml of cultured E.coli cells by centrifugation for 5min. at 3000 x g and completely remove the media by pipetting.
2. Add 250ul of PA1 to the collected cells and completely resuspend by vortexing or pipetting
3. Add 250ul of P2 and mix by inverting the tube 3-4 time, gently.
4. Add 350ul of PA3 and immediately mix by inverting the tube 3-4 time, gently.
5. Centrifuge the tubes for 10min. at 4℃, 13000 rpm in a micro-centrifuge.
6. Transfer the cleared lysate to the DNA binding column tube and centrifuge for 1min. at 13000 rpm.
7. Pour off the flow-through and re-assemble the DNA binding filter column with the 2.0ml collection tube.
8. Add 700ul of W2 to the DNA biding column tube and centrifuge for 1min. at 13000 rpm
9. Dry the column by additional centrifugation for 1min. at 13000 rpm.
10. Transfer the DNA binding filter column to the new 1.5ml micro-centrifuge tube.
11. Add 50-100ul of EA to the DNA binding filter column, and wait for at least 1min. for elution.
12. Elute the plasmid DNA by centrifugation for 1min. at 13000 rpm.
응용
Gene cloning, sequencing, Transformation, Transfection, in-vitro transcription/translation
