Endo-Free Plasmid Maxiprep Kit

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Application
DNA Purification (DNA Purif)
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Purpose The Endo-free Plasmid Maxi 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 Maxi Kit could obtain 1500 μg pure plasmids DNA from 50~200 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 50 mL Binding Columns, Endo-free Filtering Columns, 50 mL Centrifuge Tubes, Buffer RA, Buffer RB, Buffer RC, Elution Buffer, Buffer RE, Buffer NE, EFW(70% ethanol), EFW, RNase A (40 mg/mL), Handbook
Application Notes Optimal working dilution should be determined by the investigator.
Protocol 1. Pellet 100~200 mL bacterial cells cultured overnight, centrifuge at 8,500 rpm for 5 minute and discard supernatant
2. Resuspend the cell pellets in 14 mL Buffer RA (RNase A added)
3. Add 14 mL Buffer RB and reverse up and down 5~7 times gently. Incubate for 3 minutes at room temperature
4. Add 14 mL Buffer RC, reverse up and down 5~7 times gently. Add 7 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 20 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 10 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 50 mL centrifuge tube, incubate at room temperature for 5~10 minutes without covering to make sure no ethanol remain
10. Add 5 mL Elution Buffer to binding column and incubate at temperature for 2~3 minutes. Centrifuge at room temperature 8,000 rpm for 5 minutes and discard binding column (preheat the Elution Buffer to 60 °C to increase the elution efficiency )
11. Add 5 mL Buffer RE, mix and incubate at temperature for 5 minutes
12. Add 3.5 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 10 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.