Team:Valencia/Plasmid Miniprep

• Home
• Project
  • Overview
  • Our future vision
  • Genetic engineering
  • Cultures
  • Testing other microalgae
• Dry Lab
  • Introduction
  • Modeling
  • Engineering
• Human Practices
  • Activities
  • Syn(ethic) Biology -> Blog
  • Safety
• Team
  • Team description
  • Members
  • Acknowledgments
  • Gallery
• Lab Work
  • What we did
  • Submitted parts
  • Designed Biobricks
  • Notebook
  • Protocols
• Achievements
  • Achievements
  • Judging criteria
  • Collaborations

Reagents and Materials

• Solution G1 (Cell Suspension) 50mM Tris/HCl (pH 8.0); 10 mM EDTA
• Solution G2 (Cell Lysis) 200 mM NaOH; 1% SDS (w/v)
• Solution G3 (Neutralization/Binding) Contains acetate and guanidine hydrochloride.
• Solution GX (Wash, optional) Contains guanidine hydrochloride.
• Solutions G4 (Column Wash) Contains NaCl, EDTA and Tris.HCl

Protocol

1. Harvesting Bacterial Cells: E.coli cells are pelleted by centrifugation. Remove all traces of medium carefully. Then, we make sure that culture medium back-draining from the tubes wall is removed.
2. Cell Resuspending: add 250 µl of solution G1 reconstituted with RNase to the pellet and resuspend the cells (by vortexing or with a pipette) until the suspension is homogeneous.
3. Cells Lysis: add 250 µl of solution G2 and mix gently, but thoroughly, by inverting the tube several times. Do not vortex! Incubate at room temperature for 5 min.
4. Neutralization: add 350 µl of solution G3 and mix gently but thoroughly, by inverting the tube until a homogeneous suspension is obtained. Do not vortex! Centrifuge. The mixture at room temperature and at maximum speed for 10 min.
5. Column Loading: place a JETQUICK spin column into a 2 ml receiver tube (provided). Load the supernatant from step 4 into the spin column. Centrifuge at>12.000 x g for 1 min. Discard the flowthrough.
After having emptied the receiver tube re-insert the micro-apin column into it. Add 500 µl of reconstituted buffer GX into the spin column, and centrifuge at>12.000 x g for 1 min. Discard flowthrough and place the JETQUICK column back into the same receiver tube.
6. Column Washing: empty the receiver tube, and re-insert the spin column into the receiver tube. Add 500 µl of reconstituted buffer G4 and centrifuge at >12.000 x g for 1 min. Discard flowthrough and place the spin column back into the same receiver. Centrifuge again at maximum speed for 1 min.
7. Plasmid Elution: higher DNA concentrations can be obtained if the elutions is carried out in only 50 µl elution buffer volume. In this case, preheat your elution buffer to 65-70ºC, add the buffer onto the center of the silica matrix of the spin column and let stand for 1 min before centrifugation. Preheated elution buffer is generally recommended when plasmids larger 5 kb are eluted. DNA eluted in water should be stored at -20ºC. Centrifuge at >12.000 x g for 2 min.