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质粒小量提取试剂盒(柱纯化法)

 

产品名称:无内毒素质粒大量提取试剂盒

英文名称:Endo-free Plasmid Maxi Kit

产品描述:The TonkBio Endo-free Plasmid Maxi Kit is designed for efficient and convenient extraction of endo-free DNA. This system applies guanidine salt free reagent to get high pure plasimd, no salt remain, and the concentration of endotoxin below 0.1 EU/μg. 

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TB50007A

10 Reactions

 ¥1521

PDF说明书

 

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 guanidine 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 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.

Important notes before staring

  • Read handbook carefully before using this kit.
  • Add all RNase A to Buffer RA, store at 2-8℃.
  • Add 154 mL absolute ethanol to EFW(70% ethanol)according to the label and mix.
  • Check out Buffer RB to ensure no sediment found. Otherwise, heat the Buffer to 37℃ with gentle shaking.
  • All operation should done at room temperature.
  • Use endo-free pipette tips from step “11”.

Standard DNA Purification Protocol

  • Pellet 100~200 mL bacterial cells cultured overnight, centrifuge at 8,500 rpm for 5 minute and discard supernatant;
  • Resuspend the cell pellets in 14 mL Buffer RA (RNase A added);
  • Add 14 mL Buffer RB and reverse up and down 5~7 times gently. Incubate for 3 minutes at room temperature;
  • 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;  
  • Add the supernatant to Binding Column, centrifuge at room temperature 8,000 rpm for 2 minutes, discard filtrate (filter several times if necessary);
  • 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;
  • 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;
  • Place binding column back into centrifuge tube, centrifuge at room temperature 8,000 rpm for 5 minutes and discard filtrate;
  • 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;
  • 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 );
  • Add 5 mL Buffer RE, mix and incubate at temperature for 5 minutes;
  • Add 3.5 mL Buffer NE, mix and centrifuge at room temperature 10,000 rpm for 5 minutes.
  • Transfer supernatant to Endo-free Filtering Column, centrifuge at room temperature 10,000 rpm for 5 minutes and discard filtering column;
  • 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;
  • Add 10 mL EFW(70% ethanol) to wash plasmid, centrifuge at room temperature 10,000 rpm for 5 minutes and discard supernatant;
  • Repeat the step of “15”, discard supernatant as much as possible;
  • Open and incubate at room temperature for 5~10 minutes, add appropriate EFW to resuspend plasmid.

Troubleshooting guide

Low Plasmid yield

  • Bacterial culture is too old. Inoculate antibiotic-containing media with fresh isolated bacterial colony from an overnight plate.
  • Low-copy plasmids. Increase the volume of bacterial liquid.

3. Cracking is not sufficient by Buffer RB. 

Purity do 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 pulltion may cause OD260/OD280 < 1.8. And plasmid degradation or RNA pulltion may lead to OD260/OD280 > 2.0.

 

 

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