Endo-Free Plasmid Midiprep Kit

Details for Product No. ABIN5666468,
Application
DNA Purification (DNA Purif)
Options
Purpose The Endo-free Plasmid Midi Kit is designed for efficient and convenient extraction of endo-free DNA. This system applies salt free reagent to get high pure plasmid, no salt remain, and the concentration of endotoxin below 0.1 EU/μg.
Characteristics The Endo-free Plasmid Midi Kit could obtain 200 μg pure plasmids DNA from 10-50 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 15 mL Binding Columns, Endo-free Filtering Columns, 15 mL Centrifuge Tubes, Buffer RA, Buffer RB, Buffer RC, Elution Buffer, Buffer RE, Buffer NE, EFW (70% ethanol), EFW, RNase A (13 mg/mL), Handbook
Application Notes Optimal working dilution should be determined by the investigator.
Protocol 1. Pellet 10~50 mL bacterial cells cultured overnight, centrifuge at 8,500 rpm for 5 minute and discard supernatant
2. Resuspend the cell pellets in 1.6 mL Buffer RA (RNase A added)
3. Add 1.6 mL Buffer RB and reverse up and down 5~7 times gently. Incubate for 3 minutes at room temperature.
4. Add 1.6 mL Buffer RC, reverse up and down 5-7 times gently. Add 0.8 mL absolute ethanol, mix and centrifuge at 8,000 rpm for 5 minutes at room temperature
5. Add the supernatant to Binding Column, centrifuge at room temperature 8,000 rpm for 2 minutes, discard filtrate (filter several times if necessary)
6. Place binding column back into centrifuge tube, add 5 mL 80 % ethanol. Centrifuge at room temperature 8,000 rpm for 2 minutes and discard filtrate
7. Place binding column back into centrifuge tube, add 5 mL absolute ethanol. Centrifuge at room temperature 8,000 rpm for 2 minutes and discard filtrate
8. Place binding column back into centrifuge tube, centrifuge at room temperature 8,000 rpm for 5 minutes and discard filtrate
9. Place binding column back into new 15 mL centrifuge tube, incubate at room temperature for 5~10 minutes without covering to make sure no ethanol remain
10. Add 1.5 mL Elution Buffer to binding column and incubate at room temperature for 2~3 minutes. Centrifuge at room temperature 8,000 g for 5 minutes and discard binding column (preheat the Elution Buffer to 60 °C to increase the elution efficiency )
11. Add 2 mL Buffer RE, mix and incubate at room temperature for 5 minutes
12.Add 1.35 mL Buffer NE, mix and centrifuge at room temperature 10,000 rpm for 5 minutes.
13. Transfer supernatant to Endo-free Filtering Column, centrifuge at room temperature 10,000 rpm for 5 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 10,000 rpm for 15 minutes and discard supernatant
15. Add 5 mL EFW(70 % ethanol) to wash plasmid, centrifuge at room temperature 10,000 rpm for 5 minutes and discard supernatant
16. Repeat the step of ""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.