Paul.xu at 18:17, 4 November 2018

2018-11-04T18:17:42Z

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1, Spin down 2~5 ml E coli cells. 2. Add 0.25 ml P1. 3, Resuspend by votexing. 4, Add 0.25 ml P2. Lyse for 2~5 min. 5. Add 0.35 ml N3, mix 6. Spin for 2~5 min. 7. Transfer supernatant to another clean 1.5 ml microcentrifuge tube. 8. Add 0.7 vol of isopropanol (0.6 ml). 9. Spin for 3~5 min, dump off liquid. 10. Resuspend pellet in 100 ul of EB (10 mM Tris HCl, pH 8.0) 11. Add 100 ul of 2.9 mM spermidine stock. Vortex 12. Spin for 1~2 min. 13. Wash with 300 ul of 50% isopropanol solution of 10 mM MgCl2, 300 mM NaCl, and 25 mM EDTA. 14. Wash with 100 ul of 70% ethanol. 15. Remove residual ethanol 16. Dissolve pellet in 50 ul EB.

== Large Scale Purification ==

After precipitation, DNA is dissolved in EB and load to a Q Sepharose high-performance Spectrum column (2.5 cm x 60 cm) packed with 150 ml Q Sepharose high-performance and equilibrated in 10 column volumes of TE with 570 mM NaCl. Loading and elution is performed at a linear velocity of 90 cm/hr.
[[Category:Protocols]]

Plasmid Precipitation using spermine - Revision history

Paul.xu at 18:17, 4 November 2018