The BAC DNA MiniPrep Kit is designed for the rapid preparation of BACs (bacterial artificial chromosomes) and other large DNA constructs from small batch cultures of Escherichia coli. Purification of constructs up to 130 kb in size has been verified.
The DNA is preferentially purified from other cellular components such as genomic DNA and RNA. Typical DNA recoveries range between 0.6 and 1 µg from 3.0 mL of bacterial culture. The purified DNA is fully digestible with all restriction enzymes tested, and is completely compatible with manual or automated sequencing to achieve 95-100% accuracy.
BAC DNA could be purified with this kit using either an alcohol-precipitation protocol or a column-based protocol, depending on the volume of culture input and/or downstream application requirement. For all input culture volumes (up to 100 mL) and downstream applications that require intact plasmid, the alcohol precipitation protocol is to be followed. In this procedure, a series of alcohol precipitations and washes are performed to obtain clean, intact plasmid DNA.
For input culture volumes of up to 5 mL and downstream applications that are sensitive to contaminants from purification (such as PCR and sequencing), an alternative column purification protocol could be followed.
The column purification method relies on the use of spin column chromatography. Biosynthesis's resin binds DNA in a manner that depends on ionic concentrations, thus the DNA will bind to the column while most of the RNA, proteins and other contaminants will either flowthrough or be retained on top of the resin (BIND).
The bound DNA is then washed using the provided Wash Solution in order to remove any remaining impurities (WASH), and the purified plasmid DNA is eluted with the Elution Buffer (ELUTE). Refer the procedure flowchart given below.
This kit is designed to process either 50 x 1.5 mL culture samples, 30 x 5 mL culture samples, 15 x 10 mL culture samples, 6 x 40 mL culture samples or 3 x 100 mL culture samples.
Features and Benefits
- Fast and easy processing - Rapid alcohol precipitation or spin-column purification allows for the processing of multiple samples in 1 hour.
- High binding capacity of columns - The binding capacity of the columns in the BAC DNA MiniPrep Kit is 25 µg.
- High recovery of DNA - 0.6 to 1 µg of BAC DNA can be purified from 3.0 mL of bacterial culture.
- Recovered DNA is suitable for downstream applications - Purified BAC DNA is fully compatible with restriction enzyme digestions and manual or automated sequencing.
Purified BAC DNA is ready for a range of downstream applications including:
- restriction digestions