Endo-Free Plasmid Miniprep Kit

Details for Product No. ABIN5666470,
Application
DNA Purification (DNA Purif)
Options
Purpose The Endo-free Plasmid Mini Kit is designed for efficient and convenient extraction of endo-free DNA. This system applies salt free reagent to get high pure plasimd, no salt remain, and the concentration of endotoxin below 0.1 EU/μg.
Characteristics The Endo-free Plasmid Mini Kit could obtain 50 μg pure plasmids DNA from 1-4 mL culture. And the ratio of OD260/OD280 is generally between 1.8 and 2.0. The plasmid DNA could be used for PCR, enzyme digestion, sequencing, in vitro transcription and transfection.
Components 1.5 mL Binding Columns, Endo-free Filtering Columns, 1.5 mL Centrifuge Tubes, Buffer RA, Buffer RB, Buffer RC, Elution Buffer, Buffer RE, Buffer NE, EFW (70% ethanol), EFW, RNase A (8 mg/mL), Handbook
Application Notes Optimal working dilution should be determined by the investigator.
Protocol 1. Pellet 1~4 mL bacterial cells cultured overnight, centrifuge at 12,000 rpm for 1 minute and discard supernatant
2. Resuspend the cell pellets in 200 μL Buffer RA (RNase A added)
3. Add 200 μL Buffer RB and reverse up and down 5~7 times gently. Incubate for 3 minutes at room temperature
4. Add 200 μL Buffer RC, reverse up and down 5-7 times gently. Add 100 μL absolute ethanol, mix and centrifuge at 12,000 rpm for 10 minutes at room temperature
5. Add the supernatant to Binding Column, centrifuge at room temperature 12,000 rpm for 2 minutes, discard filtrate
6. Place binding column back into centrifuge tube, add 650 μL 80 % ethanol. Centrifuge at room temperature 12,000 rpm for 1 minutes and discard filtrate
7. Place binding column back into centrifuge tube, centrifuge at room temperature 12,000 rpm for 2 minutes and discard filtrate
8. Repeat step ""7""
9. Place binding column back into new 1.5 mL centrifuge tube, incubate at room temperature for 5~10 minutes without covering to make sure no ethanol remain
10.Add 200 μL Elution Buffer to binding column and incubate at temperature for 2~3 minutes. Centrifuge at room temperature 12,000 rpm for 1 minutes and discard binding column (preheat the Elution Buffer to 60 °C to increase the elution efficiency)
11.Add 200 μL Buffer RE, mix and incubate at temperature for 5 minutes
12.Add 140 μL Buffer NE, mix and centrifuge at room temperature 12,000 rpm for 2 minutes
13. Transfer supernatant to Endo-free Filtering Column, centrifuge at room temperature 12,000 rpm for 2 minutes and discard filtering column
14. Add the same volume isopropanol (or two times volume absolute ethanol), mix and incubate at temperature for 15 minutes. Centrifuge at room temperature 12,000 rpm for 15 minutes and discard supernatant
15.Add 500 μL EFW(70 % ethanol) to wash plasmid, centrifuge at room temperature 12,000 rpm for 5 minutes and discard supernatant
16. Repeat the step ""15"", discard supernatant as much as possible
17.Open and incubate at room temperature for 5~10 minutes, add appropriate EFW to resuspend plasmid.
Restrictions For Research Use only
Storage RT,-20 °C
Storage Comment Store RNase A at -20°C for one year, other system components at room temperature (15~25°C) at least for two years.