Maxiprep preparation of Plasmid DNA
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实验概要
The PureLink™ HiPure Plasmid DNA Maxiprep Kit allows purification of 500–850 μg of high-quality plasmid DNA from 100–200 mL overnight E. coli cultures in ~2 hours when cloning high copy number plasmids.
实验原理
The PureLink™ HiPure Plasmid Filter Purification Kits allow isolation of high yields of highly pure plasmid DNA. The HiPure Filter Column provides rapid clearing of the bacterial lysate without the need for centrifugation. The lysate Filtration Cartridge is integrated into the DNA Binding Column and combines the steps of clearing the bacterial lysate with binding the DNA directly to the anionexchange resin. The HiPure Filter Column protocol reduces time and effort for plasmid purification. The kits are designed to efficiently isolate plasmid DNA from E. coli in 1.5–2.5 hours using anion-exchange columns without the use of any organic solvents or cesium chloride (CsCl). The isolated plasmid DNA purity is equivalent to two passes through CsCl gradients and has low endotoxin levels.
主要试剂
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 Maxi Columns
Column Holder
PureLink™ HiPure Precipitator Module (supplied with cat. nos. K2100-26 and K2100-27 only)
Note: For Maxipreps of low copy number plasmids from bacterial cultures of >200 mL, use twice the amount of Resuspension Buffer (R3), Lysis Buffer (L7), and Precipitation Buffer (N3) as directed in the protocol.
Order the PureLink™ HiPure BAC Buffer kit for additional buffers if the buffers in the kit are insufficient for using all of the columns when following this protocol.
实验材料
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
50-mL centrifuge tubes capable of withstanding centrifugation forces >12,000 × g
Centrifuge capable of centrifuging at >12,000 × g at 4°C
The PureLink™ HiPure Plasmid DNA Maxiprep Kit allows purification of 500–850 μg of high-quality plasmid DNA from 100–200 mL overnight E. coli cultures in ~2 hours when cloning high copy number plasmids.
实验原理
The PureLink™ HiPure Plasmid Filter Purification Kits allow isolation of high yields of highly pure plasmid DNA. The HiPure Filter Column provides rapid clearing of the bacterial lysate without the need for centrifugation. The lysate Filtration Cartridge is integrated into the DNA Binding Column and combines the steps of clearing the bacterial lysate with binding the DNA directly to the anionexchange resin. The HiPure Filter Column protocol reduces time and effort for plasmid purification. The kits are designed to efficiently isolate plasmid DNA from E. coli in 1.5–2.5 hours using anion-exchange columns without the use of any organic solvents or cesium chloride (CsCl). The isolated plasmid DNA purity is equivalent to two passes through CsCl gradients and has low endotoxin levels.
主要试剂
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 Maxi Columns
Column Holder
PureLink™ HiPure Precipitator Module (supplied with cat. nos. K2100-26 and K2100-27 only)
Note: For Maxipreps of low copy number plasmids from bacterial cultures of >200 mL, use twice the amount of Resuspension Buffer (R3), Lysis Buffer (L7), and Precipitation Buffer (N3) as directed in the protocol.
Order the PureLink™ HiPure BAC Buffer kit for additional buffers if the buffers in the kit are insufficient for using all of the columns when following this protocol.
实验材料
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
50-mL centrifuge tubes capable of withstanding centrifugation forces >12,000 × g
Centrifuge capable of centrifuging at >12,000 × g at 4°C
