CopyControl™ BAC Cloning Kits (BamH I, EcoR I, or Hind III)
CopyControl™ pCC1BAC™ Cloning-Ready Vectors (BamH I, EcoR I, or Hind III)

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Applications

• Preparation of BAC libraries that can be maintained at single copy number and induced to high-copy number as needed.

CopyControl™ BAC Cloning Kits* are complete kits containing all molecular biology reagents needed for constructing high-quality, large-insert Bacterial Artificial Chromosome (BAC) libraries. Each kit has enough reagents to make a complete human genome size library at 10X coverage. The kits feature the CopyControl™ pCC1BAC™ Vector which contains the E. coli F-factor single-copy origin of replication and oriV, a second origin of replication, which can be used to selectively induce to high copy number.

To maintain stability, CopyControl BAC clones are grown at very low copy number during the initial cloning and screening processes. Then, immediately before DNA purification, clones can be induced to 10-20 copies per cell within 2 hours by adding CopyControl Induction Solution to the culture, for higher yields of BAC DNA. The short, 2-hour induction of CopyControl BAC clones to high-copy number does not decrease their stability, based on analysis of Hind III restriction patterns of a large number of induced versus uninduced clones that ranged in size up to ~200 kb. Alternatively, the CopyControl BAC Autoinduction Solution can be used. Autoinduction Solution is added to the culture medium prior to inoculation, and also increases the cell growth leading to higher yield than standard Induction Solution. CopyControl BAC Cloning Kits reduce the time and labor required to construct a BAC library.

Phage T1-resistant TransforMax™ EPI300™-T1^R Electrocompetent E. coli cells, or standard TransforMax™ EPI300™ Electrocompetent E. coli cells, which are required for induction of CopyControl BAC clones to 10-20 copies per cell, are available separately.

Figure 1. DNA from induced (+) and uninduced (-) cultures of 4 CopyControl™ BAC clones. An equal number of cells were processed from both induced and uninduced cultures. Samples were resolved on a 0.8% agarose gel and stained with SYBR® Gold (Invitrogen). The induced cultures yielded >15-fold more CopyControl BAC clone DNA. M=BAC-Tracker™ Supercoiled DNA ladder.

Figure 2. Not I digestion of 2 µl of DNA from induced (+) and uninduced (-) cultures of 4 CopyControl™ BAC clones. After resolving by PFGE and staining with SYBR® Gold (Invitrogen), clone sizes were: BAC1=130 kb, BAC2=125 kb, BAC3=140 kb and BAC4=145 kb. M=Pulsed-Field Gel Marker.

*Covered by issued and/or pending patents.
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