NucleoBond® BAC 100

Principle

The NucleoBond BAC 100 Kit is designed for purifying large DNA fragments such as cosmids, bacteriophage P1 clones, P1-derived artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs), without phenol/chloroform extraction. The procedure takes less than 1 hour to isolate BAC DNA free from any kind of RNA, proteins, metabolites, dyes, or carbohydrates. After purification, all nucleic acids show absorbance ratios of A_260/A280 as high as 1.8–2.0.

Low amounts of impurities, which often act as strong inhibitors for enzymatic processing, sequencing, transfections, transcriptions, etc., are removed completely.

NucleoBond Folded Filters, which are specially designed to eliminate the centrifugation step after the alkaline lysis step, are provided with the kit. Using the filters reduces hands-on-time and eliminates plastic waste common to other filtration systems. In approximately 10 minutes of unattended operation, complete removal of SDS and cellular debris from plasmid samples is achieved. NucleoBond Folded Filters do not induce shearing of large DNA constructs, such as PACs or BACs.

The patented anion exchange resin in the NucleoBond column is known to provide high-purity DNA equivalent or superior to that obtained by two successive rounds of CsCl ultracentrifugation. The plasmid purification procedure is performed in the absence of toxic material such as phenol, chloroform, ethidium bromide, or CsCl. 

Procedure for NucleoBond 100 BAC Purification

Bacteria harvested from the culture broth are lysed by using a modified alkaline/SDS procedure. The bacterial lysate is cleared by filtration and loaded onto the equilibrated column, where plasmid DNA binds to the anion exchange resin. After subsequent washing steps, the purified plasmid DNA is eluted in a high-salt buffer and precipitated with isopropanol. The plasmid DNA is reconstituted in TE buffer for further use.

High-Throughput BAC Purification

Preparation of a BAC clone (163 kb insert) using the NucleoBond BAC 100 kit. The purified BAC clone was analyzed by autoradiographic sequencing (Panel A) and restriction analysis (Panel B).

Data was kindly provided by Universität Gießen, Inst. für Med. Mikrobiologie, Gießen, Germany.

Data was kindly provided by Universität Gießen, Inst. für Med. Mikrobiologie, Gießen, Germany.