Endotoxin-free Plasmid DNA Mini-prep Kit, 50 T (for Nucleic Acid Extraction)

Product Information

Description/Introduction

The kit uses a specially formulated buffer solution, combined with the reversible adsorption technology of nucleic acid by silica gel membrane, can almost completely remove endotoxin, protein and other impurities from 1~5 mL Escherichia coli culture, and the yield of plasmid DNA is 5~20 μg. The extracted plasmid can be used in biological experiments such as restriction enzyme digestion, ligation reaction, PCR amplification, sequencing, transformation, transfection, etc.

Storage and Shipping Conditions

RNase A is shipped with wet ice and stored at -20°C. Other reagents are shipped and stored at room temperature; valid for 12 months.

Product Contents

Before starting (please read carefully)

1. Anhydrous ethanol and Nuclease-free 1.5 mL centrifuge tube are required but not supplied in this kit.

2. Prepare 42 water bath or heating module.

3. Add all the RNase A provided in the kit to Buffer P1 before use, and it can be stored at 4°C for 6 months.

4. Please add 60 mL anhydrous ethanol to Buffer PW before use.

5. If Buffer P2 precipitates, please heat it in a water bath at 37°C for a few minutes to restore clarification. After using Buffer P2, the lid should be closed immediately to avoid long-term contact with air.

6. Please pre-cool Buffer P3 at 4°C before use.

Assay Protocol / Procedures

1. Column balance: Add 500 μL Buffer BL to HiBind DNA Mini Column (HiBind DNA Mini Column is put into Collection Tube ahead of time), centrifuge at 12,000 rpm for 1 min at room temperature, discard the filtrate, and put the HiBind DNA Column back into the Collection Tube (the treated column is best used immediately).

2. Harvest bacterial culture from 1~5 mL fresh bacterial fluid by centrifugation at 12,000 rpm for 1 min at room temperature (remove the supernatant as much as possible).

3. Add 250 μL Buffer P1 (please check whether or not to add the RNase A first), use a pipette or vortex oscillator to thoroughly suspend the bacteria (be sure to disperse the bacteria thoroughly, otherwise it will affect the lysis, resulting in low quality and purity of the extracted plasmid).

4. Add 250 μL Buffer P2, immediately and gently turn it upside down for 8~10 times to make the bacteria fully lysed (this step should not be violently shaken, and should be done within 5 min). At this point, the solution should become clear and sticky, if it does not become clear, it may be that there are too many bacteria and the lysis is not complete, and consideration should be given to reducing the amount of bacteria.

5. Add 250 μL Buffer P3 (pre-cool in advance), immediately and gently upside down 10~12 times, the solution appears compact agglomerate, centrifuge at 12,000 rpm for 10 min at room temperature, Transfer the supernatant to a new 1.5mL centrifuge tube.

6. Add 75 μL Buffer ER, place 10 min on the ice after mixing upside down (mix upside down 3~5 times during the period), the solution is transparent blue.

7. Incubate at 42°C for 5 min, the solution recovers turbidity. Centrifuge at 12,000 rpm for 10 min at room temperature, the solution is divided into upper and lower layers, the upper layer is clear water phase and the lower layer is blue. Carefully collect the upper water phase into the new Nuclease-Free 1.5 mL centrifuge tube.

8. Add anhydrous ethanol 0.5 times the volume of the above solution, mix upside down and transfer to HiBind DNA Mini Column (no more than 700 μL each time). Centrifuge at 12,000 rpm for 1 min at room temperature, discard the filtrate in the Collection Tube. Put the HiBind DNA Mini Column back into the Collection Tube (the solution can pass through the column many times).

9. Add 600 μL Buffer ED to the HiBind DNA Mini Column, centrifuge at 12,000 rpm for 1 min at room temperature, discard the filtrate in the Collection Tube. Put the HiBind DNA Mini Column back into the Collection Tube.

10. Add 600 μL Buffer PW to HiBind DNA Mini Column, centrifuge at 12,000 rpm for 1 min at room temperature, discard the filtrate in the Collection Tube. Put the HiBind Mini DNA Column back into the Collection Tube.

11. Repeat step 10.

12. Centrifuge at 12,000 rpm for 2 min at room temperature, place the HiBind DNA Mini Column in a new Nuclease-Free 1.5 mL Centrifuge Tube. Open the lid and place 5 min at room temperature to volatilize the ethanol thoroughly.

13. Add 50 μL Buffer TE or Endo-Free Water to the membrane center of the HiBind DNA Mini Column and place it at room temperature for 2 min. Centrifuge at 12,000 rpm for 2 min. If you need to increase the efficiency of plasmid recovery, the obtained solution can be re-added to HiBind DNA Mini Column, place at room temperature for 2 min and centrifuge at 12,000 rpm for 2 min.

14. The obtained plasmid DNA can be preserved for a long time at -20°C.

Note

1. Please read the Product Manual carefully before use.

2. When the copy number of the extracted plasmid is low or the plasmid fragment is larger than 10kb, the amount of bacteria collection should be increased, and the dosage of Buffer P1, Buffer P2 and Buffer P3 should be increased in equal proportion.

3. Ethanol should be completely volatilized before eluting the plasmid to avoid the influence of residual ethanol on downstream experiments.

4. Buffer TE can be preheated at 60~65°C and the elution incubation time can be prolonged to improve the elution efficiency.

5. For your safety and health, please wear a lab coat and disposable gloves.

For Research Use Only!

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