The Nucleogen Plasmid Purification Midi Kit for Transfection is designed for isolation of transfection grade plasmid, cosmid DNA or BAC DNA for use in routine molecular biology applications such as transfection, Microinjection, In vitro transcription, fluorescent and radioactive sequencing and cloning. Versatile Nucleogen Plasmid spin columns can be used either in centrifuges or on vacuum manifolds. The vacuum procedure provides simplified handling and faster sample processing. Nucleogen Plasmid spin columns can be vacuum processed using the commercial manifold with luer connectors.

Principle

Nucleogen Cartridges are special filter units designed to replace the centrifugation step following alkaline lysis of bacterial cells. (UltraHiFast and UltraFast)

Nucleogen Plasmid Midi columns contain a unique silica gel membrane that binds up to DNA in the presence of a high concentration of chaotropic salt, and allows elution in a small volume of low-salt buffer. Nucleogen Plasmid membrane technology eliminates time consuming phenol–chloroform extraction, as well as the problems and inconvenience associated with loose resins and slurries.

Precipitator module replaces the centrifugation step traditionally used to collect isopropanol-precipitated DNA following purification. The Precipitator module traps the precipitated DNA while the isopropanol–buffer mixture flows through. The DNA is then simply eluted from the Precipitator into a microcentrifuge tube with TE buffer or water. This unique module also eliminates the risk of pellet loss, which can occur during decanting of the supernatant following centrifugation. (UltraHiFast)


Procedure

Neutralized bacterial lysates are cleared by Catridge (UltraHiFast and UltraFast). The cleared lysate is applied to a Midi column for plasmid DNA purification. Eluted DNA is mixed with isopropanol and applied to the Precipitator module using the syringe provided (UltraHiFast). The concentrated and desalted DNA is then eluted from the Precipitator directly into a microcentrifuge tube with TE buffer or water.

Applications

Nucleogen Plasmid Purification Midi Kits for Transfection provide reproducible yields of transfection grade DNA suitable for use in most applications, including:

Transfection of endotoxin-sensitive cells
Cloning to Viral Backbone
Establish Stable Cell Line
Automated fluorescent sequencing, including capillary sequencing
Radioactive sequencing
Restriction digestion
Screening
Microinjection
In vitro transcription
PCR template preparation
All other exzymatic reactions

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1,3,5 and 7 lanes : Q company Midi Kit
2,4,6 and 8 lanes : Nucleogen Plasmid Midi Kit
1 and 2 lanes : pLenti6-U6-RGS1-shRNA (6726 bp)
3 and 4 lanes : pLenti6-H1TO-Gas-shRNA (6728 bp)
5 and 6 lanes : pLP/VSVG (5821 bp)
7 and 8 lanes : p3X-FLAG-CMV10 (6299 bp)  

 Transfection efficiency test
 Test Cell Line: 293
 Test plasmid: pEGFP
 GFP positive cells : 95%

Kit Contents
UltraHiFast	Midi-columns, Cartridge, Precipitator, Syringe, Cell Suspension Solution, Cell Lysis Solution, Neutralization Solution, Wash Solution, Elution Buffer, TE Buffer, RNase A
UltraFast	Midi-columns, Cartridge, Cell Suspension Solution, Cell Lysis Solution, Neutralization Solution, Wash Solution, Elution Buffer, TE Buffer, RNase A
Fast	Midi-columns, Cell Suspension Solution, Cell Lysis Solution, Neutralization Solution, Wash Solution, Elution Buffer, TE Buffer, RNase A