Cyclo-PrepTM K179 - Miniprep Plasmid DNA Purification Kit

Directions for use
Product Description:

Cyclo-Prep is a spin column-based kit for rapid purification of plasmid DNA from 1-2 ml liquid culture. In just 15 minutes, prepare ultra-pure plasmid DNA (A260/A280 ( 1.8) with a yield of 2-15 µg per ml of E. coli culture using your tabletop microcentrifuge. The DNA isolated from Cyclo-Prep is suitable for automated fluorescent sequencing, PCR, in vitro transcription, transformation and restriction enzyme digestion. The simple protocol provides an environmentally safe, convenient, and fast alternative to common phenol-based DNA purification procedures.

 
Kit Components:
The Cyclo-Prep Kit contains reagents sufficient for 50 minipreps. All reagents can be stored at room temperature. Each kit includes the following components:

  • Solution 1     10ml

  • Solution 2     10ml

  • Solution 3     10ml

  • Wash Solution (with ethanol) 80ml

  • Cyclo-Prep Spin Columns    50 each

  • Collection Tubes                  50 each

Required Equipment: A micropipettor, bench-top microcentrifuge, and 1.5 - 1.7 ml microfuge tubes.

Protocol for Plasmid DNA Purification:

  • Transfer 1-2 ml of an overnight culture to a microcentrifuge tube. Pellet the bacterial cells by spinning at top speed (12-14,000 x g) for 30 sec. to 1 min. Remove the supernatant.

  • Add 200 µL of Solution 1 to the pellet of cells. Pipette up and down (or vortex) to achieve complete resuspension.

  • Add 200 µL of Solution 2. Mix by gentle inversion of the tube 5-6 times.

  • Add 200 µL of Solution 3. Mix by gentle inversion of the tube 5-6 times.

  • Pellet the precipitate by centrifugation (12-14,000 x g) for 5 min. A white pellet will form at the bottom or along the side of the microcentrifuge tube.

  • Place a Cyclo-Prep Spin Column into a Collection Tube. Carefully remove the clear lysate from Step 6 and add it directly to the spin column.

  • Spin (12-14,000 x g) for 30 sec. Remove the spin column from the collection tube and discard the filtrate. The same collection tube can be used for the following step.

  • Add 700 µL of the Wash Solution to the spin column and spin (12-14,000 x g) for 30 sec. Remove the spin column from the collection tube and discard the filtrate. Spin for an additional 3 min. (12-14,000 x g) to remove residual traces of ethanol.

  • Remove the spin column and place it in a new microcentrifuge tube (not provided). Add 50-100 (l of preheated (60-70°C ) H2O or TE Buffer.

  • Elute the plasmid DNA by centrifugation (12-14,000 x g) for 30 sec.1, 2 Store the eluted DNA at -20(C

1. To increase the yield of DNA by as much as 10-15%, Steps 9-10 can be repeated.

2. For automated fluorescent sequencing, an ethanol precipitation step is recommended following elution.

 

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Interbiotech

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