Principle / Procedure

High selectivity and specific characteristics of the optimal silica matrix make this anion exchanger ideal for the purification of very large constructs, such as P1, BACs, and PACs. Using the NucleoBond® Xtra BAC kit, it takes just 75 min to isolate, for example, BAC DNA free from any kind of RNA, proteins, metabolites, dyes, or carbohydrates. Low amounts of impurities, which often act as strong inhibitors for enzymatic processing, transfections, transcriptions, etc., are completely removed.

LyseControl — for highest yields

The new LyseControl, included in the NucleoBond® Xtra BAC procedure, visualizes a complete and efficient alkaline lysis. LyseControl is a blue reagent premixed for your convenience with Lysis Buffer LYS-BAC. Upon addition of lysis buffer to the resuspended cells (Fig. A), the cell suspension turns blue (Fig. B). The blue solution is gently inverted up to 5 times and incubated for 5 min to avoid contamination with genomic DNA. Upon addition of Buffer NEU-BAC (Fig. C) to neutralize the lysate, LyseControl turns colorless. Traces of blue LyseControl (Fig. D) indicate an insufficient mixing. Sufficient mixing and complete neutralization is needed to renature plasmid DNA, precipitate proteins / gDNA, and remove SDS, which inhibits plasmid binding.
The solution has to be inverted until a homogeneous and 100 % colorless solution (Fig. E) is achieved. A gentle inverting is highly recommended to prevent genomic DNA contamination. LyseControl helps to verify a completed alkaline lysis providing optimal yields.

Application data

Highest yield in less time – comparison to competitor kits

BAC DNA (300 kbp) was isolated in triplicate from 500 mL E. coli DH5a using NucleoBond® Xtra BAC and competitor products (Q and I). After precipitation, BAC DNA was reconstituted in 1000 μL TE buffer and 5 μL of each sample was used for analysis on a 1 % TAE agarose gel.
Obtained yields: MN: 150 μg, Q: 44 μg, I: 75 μg.

Suitable for restriction digestion

Restriction digestion of BAC DNA isolated with NucleoBond® Xtra BAC. BAC DNA samples were purified from overnight cultures of E. coli DH5a transformed with a pBAC10 clone. Approximately 3 μg of DNA from each sample was digested with 3 units of MspI, HindIII, or EcoRI at 37 °C for 2 hours. Digestions were analyzed on a 1 % TAE agarose gel. Lane 1: undigested BAC DNA, lane 2: MspI digested, lane 3:
HindIII digested, lane 4: EcoRI digested, lane M: marker.

 

High speed BAC purification – comparison to competitor kits