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Endo-Free Plasmid Midiprep Kit 

The TonkBio 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.
Cat. No. Size Price Quantity  
TB50008A 20 Rxns USD 179
Tel: +1 (973) 789-8318Email: [email protected]

Components

Endo-free Plasmid Midi Kit 20 preps
15 mL Binding Columns 10×2
Endo-free Filtering Columns 10×2
15 mL Centrifuge Tubes 10×2
Buffer RA 65 mL
Buffer RB 65 mL
Buffer RC 65 mL
Elution Buffer 60 mL
Buffer RE 60 mL
Buffer NE 45 mL
EFW(70% ethanol) 66 mL
EFW 12 mL
RNase A (13 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 Midi Kit is designed for efficient and convenient extraction of endo-free DNA. This system applies salt free reagent to get high purity plasmid, no salt remain, and the concentration of endotoxin below 0.1 EU/μg.

The TonkBio 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.

Purification 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℃ 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.

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 154 mL absolute ethanol to 70% Endo-free ethanol and mix well.
  • 4.Check out Buffer RB to ensure no sediment found. Otherwise, heat the Buffer to 37℃ with gentle shaking.
  • 5.All operation should 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. Cracking is not sufficient 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. Mix immediately 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 Midiprep Kit

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