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 A260/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 b 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
The NucleoSpin 96 Flash Kit lets you rapidly isolate BAC DNA from bacterial cultures using a high-throughput method. Process samples in a 96-well format using either a vacuum manifold or a tabletop centrifuge. Obtain BAC yields of <1 µg from 1.3–3.9 ml of E. coli culture.
Application Data for NucleoBond 100 BAC Purification
Preparation of a BAC clone 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. Restriction Analysis of Purified BAC Clones Five different BAC clones (in pBeloBAC2, containing 50–100 kb inserts) were each grown in 200 ml LB medium and purified using NucleoBond BAC 100 kits, yieldin approximately 50–70 µg DNA per prep. For analysis, 0.5 µg of each purified construct was digested with EcoRI. Undigested and EcoRI-digested samples were electrophoretically separated on 0.8% agarose gels, in 1X TAE. Lanes 1: Molecular weight marker (1 Kb Standard, MBI Fermentas) Lanes 2: 0.5 µg BAC DNA
Lanes 3: 0.5 µg BAC DNA (EcoRI-digested) 
Purification and Subsequent Restriction Analysis of a BAC Clone The BAC construct pBeloBAC11 was purified using the NucleoBond BAC 100 kit. The high quality of the purified construct was demonstrated by the distinct band(s) seen after analysis of an undigested sample (Panel A, Lane 2) and an EcoRI-digested sample (Panel B, Lane 2) on a 0.8% agarose gel. In contrast, EcoRI-digested chromosomal DA (Panel B, Lane 3) displayed a “smear” and no distinct fragments. Panel A. Agarose Gel Analysis of an Undigested Purified BAC Lane 1: Molecular weight marker (1 Kb Standard, MBI Fermentas) Lane 2: 500 ng of purified pBeloBAC11 (~100 kb) Panel B. Agarose Gel Analysis of a Purified BAC and Chromosomal DNA after EcoRI Digestion Lane 1: Molecular weight marker (1 Kb Standard, MBI Fermentas) Lane 2: EcoRI digestion of purified pBeloBAC11
Lane 3: EcoRI digestion of chromosomal DNA 
Data was kindly provided by Universität Gießen, Inst. für Med. Mikrobiologie, Gießen, Germany. | 