Sample Preparation |
Purification of Cloned DNA
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Applications
- Isolation of exceptionally pure BAC DNA that can be used for many applications
including sequencing, fingerprinting, PCR, and preparation of shotgun libraries.
The BACMAX™ DNA Purification Kit was developed for easy, reliable isolation
of high-quality BAC DNA. The scalable protocol is based on a modified alkaline-lysis
procedure that typically yields 0.6-25 µg of BAC DNA from 1.5-100 ml of
a single-copy fosmid culture. Selective precipitation steps and the incorporation
of EPICENTRE's RiboShredder™ RNase Blend effectively remove contaminants
that degrade DNA and interfere with downstream applications.
The BACMAX Kit is ideal for the purification of induced BACs produced with
CopyControl™ BAC Vectors. The CopyControl cloning technology enables the
user to grow the BAC clones at single copy to ensure insert stability and cloning
of toxic gene products and then induce the clones to high-copy number for high
yields of DNA. Using the BACMAX Kit, up to 4 µg of BAC DNA can be isolated
from 1.5 ml of an induced clone.
Benefits
- Simple protocols isolate up to 0.6, 15, or 25 µg of BAC DNA from 1.5-,
40- or 100-ml cultures of a single-copy BAC, respectively.
- The kit does not use columns or binding resins that lower yields and shear
the DNA.
- Isolate the amount of DNA that is needed, without wasting kit reagents.
- Ideal for isolating DNA from single-copy BAC clones or induced CopyControl™ BAC
clones.
- No toxic organic solvents.
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Figure 1. Obtain higher yields of BAC DNA with the BACMAX™ DNA
Purification Kit. DNA was isolated from 1.5 or 3.0-ml cultures of a 153-kb
single-copy BAC clone and quantitated by fluorometry with Hoechst 33258 dye.
Culture volumes were those recommended by the Suppliers. |
Panel A |
Panel B |
Figure 2. Panel A. The BACMAX™ DNA Purification Kit consistently
yields high-quality BAC DNA that can be used directly for downstream applications
like fingerprinting. BAC DNA isolated from 10 different clones (Lanes
1 to 10) was digested with Hind III and analyzed by agarose gel electrophoresis.
Lane M, kb ladder. Panel B. BAC DNA isolated with the BACMAX™ Kit
shows no degradation after being stored for 1 year at –20°C. DNA
isolated from a 153-kb and a 165-kb BAC clone was immediately digested with Hind
III (Lane 1, 153 kb and Lane 2, 165 kb) or stored for 1 year at -20°C
and then digested with Hind III (Lane 3, 153 kb and Lane 4, 165 kb).
Samples were analyzed by agarose gel electrophoresis. Lane M, kb ladder. |
BAC-Tracker™ Supercoiled DNA Ladder
CopyControl™ BAC Cloning Kits (BamH I, EcoR I, or Hind III)
CopyControl™ pCC1BAC™ Cloning-Ready Vectors (BamH I, EcoR I, or Hind III)
EZ-Tn5™ <oriV/KAN-2> Insertion Kit
Lambda Terminase
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