Miniprep Protocol
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Bench protocol (rapid protocol) for GeneAll® Hybrid-Q™ Plasmid Rapidprep.
This protocol is designed for purification of up to 30 µg of high-copy plasmid DNA from 1 to 3 mL of o/n E. coli culture in LB medium. - Pellet 1~3 ml of culture by centrifugation
- Resuspend in 170 µl of buffer S1
- Add 170 µl of buffer S2 and mix by inverting
- Add 250 µl of buffer G3 and mix by inverting
- Transfer the lysate to EzClear™ column stack by decanting
- Centrifuge for 30 sec and discard the EzClear™ filter (upper, violet)
- (Optional) Add 500 µl of buffer AW and centrifuge for 30 sec
- Add 700 µl of buffer PW and centrifuge for 30 sec
- Centrifuge for additional 1 min
- Apply 50 µl of buffer EB and centrifuge for 1 min
The preparation time is <10 minutes. The typical yield with GeneAll® Hybrid-Q™ Plasmid Rapidprep and an elution volume of 50 µl is 5 - 6 µg of high-copy-number plasmid DNA per ml of bacterial culture (see fig. 1). The binding capacity of one column is 30 µg, thats two times more than with miniprep spin columns from other suppliers. Please compare the price of GeneAll® Hybrid-Q™ Plasmid Rapidprep to miniprep kits from other suppliers by your own – its worth doing so.
Quality Control of GeneAll® Hybrid-Q™ Plasmid Rapidprep: Restriction enzyme assay, gene cloning, PCR amplification assay and automated sequencing analysis are carried out carefully from lot to lot as quality control, and only qualified lots are shipped to customers.
Download your miniprep handbook detailing the rapid, standard and low copy protocols.
GeneAll® Hybrid-Q™ Plasmid Rapidprep Order Information
More DNA and RNA preparation and purification kits from GeneAll.
Alternatively you may follow the classical miniprep protocol. It takes up more of your time, can lead to loss of DNA and impure preparations and its a little bit cheaper, as long as one ignores the cost of waste disposal (chloroform and phenol waste needs special treatment):
Classical Mini-prep Protocol Centrifigation steps are performed at ~12,000 g. - Grow ~5 mL bacterial colonies overnight in LB medium.
- Take out 1.5 ml of bacteria culture and spin down for 30 seconds at 4 °C. Remove supernatant. Store the remainder of the culture at 4 °C.
- Add 100 µl Solution 1 (GET Buffer). Vortex to resuspend pellet completely.
- Add 200 µl of freshly prepared Solution 2.
- Close the tube tightly, and mix the contents by inverting the tube rapidly five times, store the tube on ice, and wait 2 minutes.
- Add 150 µl of ice-cold Solution 3. Close the tube and vortex it gently in an inverted position for ten seconds. Store the tube on ice for 5 minutes.
- Add 150 µl Phenol/Chloroform/Isoamyl alcohol. Vortex 5-10 seconds.
- Spin 2 minutes at 4 °C.
- Remove the supernatant and transfer it to a new tube.
- Add 2.5X volume of ethanol. Invert to mix and allow the mixture to stand for 2 minutes at room temperature.
- Spin for 5 minutes at 4 °C. You should see a white pellet now.
- Discard supernatant, e. g. by gentle aspiration and removal of adhering drops of fluid.
- Add 1000 µl 70% ethanol at 4 °C. Spin 2 min, (RT or 4 °C) and remove supernatant (as in the step before)
- Dry pellet (10+ minutes).
- Resuspend in 50-100 µl TE (pH 8.0), vortex and store DNA at -20 °C.
Solution 1 (GET Buffer) Solution 2 Solution 3
The preparation time is about 30 minutes. The typical yield of high-copy-number plasmids, prepared by this method is 3-5 µg of DNA per ml of bacterial culture.
Reference
Modified after Sambrook, Fritsch, Maniatis "Molecular Cloning, A Laboratory Manual", 2nd edition, Cold Spring Harbor Laboratory Press, New York, 1989.
GeneAll® Hybrid-Q™ Plasmid Rapidprep Order Information
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