Midiprep preparation of Plasmid DNA

实验概要

The PureLink™ HiPure Plasmid DNA Midiprep Kit allows purification of 100–350 μg of high-quality plasmid DNA from 15–25 mL overnight E. coli cultures in ~2 hours when cloning high copy number plasmids.

主要 试剂

Before Starting
Before beginning, verify that the Resuspension Buffer (R3) contains RNase A, and no precipitate has formed in the Lysis Buffer (L7).
Materials Needed
Overnight culture of transformed E. coli cells
Isopropanol
70% ethanol
Sterile, microcentrifuge tubes
PureLink™ Nucleic Acid Purification Rack
Tubes or centrifuge bottles for harvesting cells
Centrifuge and rotor appropriate for harvesting cells
Appropriate 15-mL centrifuge tubes capable of
withstanding centrifugation forces >12,000 × g
Centrifuge capable of centrifuging at >12,000 × g at 47°C
Optional : PureLink™ HiPure Precipitator Module
Components Supplied with the Kit
Resuspension Buffer (R3) with RNase A
Lysis Buffer (L7)
Precipitation Buffer (N3)
Equilibration Buffer (EQ1)
Wash Buffer (W8)
Elution Buffer (E4)
TE Buffer (TE)
HiPure Filter Midi Columns
Column Holder
Note: The protocol for the Midiprep kit of the PureLink™ HiPure Plasmid Filter Purification Kits is designed for purification of both high and low copy number plasmids without the need for adjusting buffer volumes.