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Endo-Free Plasmid Miniprep Kit 

The TonkBio 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.
Cat. No. Size Price Quantity  
TB50009A 50 Rxns USD 70
TB50009B 200 Rxns USD 219
Tel: +1 (973) 789-8318Email: [email protected]

Components

Endo-free Plasmid Mini Kit 50 preps
1.5 mL Binding Columns 50
Endo-free Filtering Columns 50
1.5 mL Centrifuge Tubes 50
Buffer RA 20 mL
Buffer RB 20 mL
Buffer RC 20 mL
Elution Buffer 12 mL
Buffer RE 12 mL
Buffer NE 10 mL
EFW(70% ethanol) 18 mL
EFW 12 mL
RNase A (8 mg/mL) 550 μL
Handbook 1

*User should prepare absolute ethanol, 80% ethanol (V/V),  isopropanol and endo-free pipette tips.

Storage and Stability

Store RNase A at -20℃ for one year, other system components at room temperature (15~25℃) at least for two years

Description

The TonkBio 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.

The TonkBio 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.

Purification 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 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.
  • Important Notes

    Notes

    • 1. Read handbook carefully before using this kit.
    • 2. Add all RNase A to Buffer RA, store at 2-8℃.
    • 3. Add 42 mL absolute ethanol to EFW (70% ethanol) and mix.
    • 4. Check out Buffer RB to ensure no sediment found. Otherwise, heat the Buffer to 37℃ with gentle shaking.
    • 5. All operation should be done at room temperature.
    • 6. Use endo-free pipette tips from step 11.

    Troubleshooting Guide

    1) Low Plasmid Yield

    1. Bacterial culture is too old. Inoculate antibiotic-containing media with freshly isolated bacterial colony from an overnight plate.
    2. Low-copy plasmids. Increase the volume of bacterial liquid.
    3. Insufficient cracking by Buffer RB.

    2) Plasmid Purified Does Not Perform Well

    1. RNase A is not added into Buffer RA or Buffer RA (RNase A added) is not storage at 4℃.
    2. Shaking violently after adding Buffer RB may break the genomic DNA.
    3. Protein is not removed clearly,immediate mixing is required after adding Buffer RC.
    4. The ratio of OD260/OD280 is generally between 1.8 and 2.0. Protein pollution may cause OD260/OD280<1.8. And plasmid degradation or RNA pollution may lead to OD260/OD280>2.0.

Datasheet Download

Endo-Free Plasmid Miniprep Kit

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