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Endo-Free Plasmid Maxiprep Kit 

The TonkBio 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.
Cat. No. Size Price Quantity  
TB50007A 10 Rxns USD 169
Tel: +1 (973) 789-8318Email: [email protected]

Components

Endo-free Plasmid Maxi Kit 10 preps
50 mL Binding Columns 10
Endo-free Filtering Columns 10
50 mL Centrifuge Tubes 20
Buffer RA 220 mL
Buffer RB 220 mL
Buffer RC 220 mL
Elution Buffer 60 mL
Buffer RE 60 mL
Buffer NE 45 mL
EFW(70% ethanol) 66 mL
EFW 30 mL
RNase A (40 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 Maxi Kit is designed for efficient and convenient extraction of endo-free DNA. This system applies guanidin salt free reagent to get high purity plasimd, no salt remain, and the concentration of endotoxin below 0.1 EU/μg.

The TonkBio 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.

Purification 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℃ 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.

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 EFW (70% ethanol) according to the label 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 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. 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. 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

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