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CopyControlTM, EpiFOSTM, MaxPlaxTM

Cloning, Genomic

CopyControl™ Fosmid Library Production Kit
CopyControl™ HTP Fosmid Library Production Kit

Applications
  • Preparation of complete and unbiased fosmid libraries, from any biological sample, that can be maintained at single-copy number and induced to high-copy number as needed, using the CopyControl™ Autoinduction Solution.
  • Construction of metagenomic libraries from microbes present in environmental samples such as water and soil.

The CopyControl™ Fosmid Library Production Kits* provide an efficient and improved method for constructing a library of approximately 40 kb clones. The CopyControl pCC1FOS™ Vector contains both the E. coli F-factor single-copy origin of replication and the inducible high-copy oriV (Fig. 1). CopyControl Fosmid clones are typically grown at single copy to ensure insert stability and successful cloning of encoded and expressed toxic protein and unstable DNA sequences. The CopyControl Fosmid clones can then be induced up to 50 copies per cell immediately before DNA purification. This step greatly increases DNA yields, while maintaining the stability of the plasmid.

The CopyControl HTP Fosmid Library contains the pCC2FOS™ Vector which is designed to optimize end-sequencing results, especially in a high-throughput setting. The primer cassette, engineered in conjunction with Agencourt Bioscience Corporation, eliminates wasteful and expensive vector sequence reads by having the 3´ ends of the primer-annealing sites only three bases from the vector/insert junction. In addition, the seven-base sequence at the 3´ end of each primer was specifically designed to minimize mispriming to any contaminating E. coli DNA present after template purification (Fig. 2).
The kit uses a strategy of cloning blunt-ended DNA fragments generated by random shearing of the DNA, to produce more complete and unbiased genomic libraries than can be obtained by partial restriction endonuclease digests. Genomic DNA is first sheared into approximately 40-kb fragments. The sheared DNA is end-repaired to generate blunt, 5´-phosphorylated ends and then size-selected by and recovered from a low-melting-point agarose gel. Finally, the size-selected DNA is ligated into the cloning-ready CopyControl pCC1FOS or pCC2FOS Vector, packaged using ultra-high efficiency MaxPlax™ Lambda Packaging Extracts (>109 pfu/µg DNA), included in the kit, and plated on the supplied TransforMax™ EPI300™ E. coli (Fig. 3).

Benefits
  • CopyControl pCC1FOS and pCC2FOS Vectors are supplied Cloning-Ready: linearized, dephosphorylated, purified, and ready for ligation.
  • No need for partial restriction endonuclease digests or pulse field gel electrophoresis to prepare the genomic DNA for cloning.
  • Maximize high-throughput end-sequence results using the pCC2FOS Vector (Fig. 4).
  • Clones can be induced from single copy up to 50 copies per cell (Fig. 5). Safely obtain higher DNA yields while maintaining the stability of single-copy-number clones.
  • High-efficiency lambda packaging eliminates background and false positives.
  • Faster and easier than BAC cloning.
i_copycontrol_vectormap.gif
Figure 1. CopyControl™ Vector map. The CopyControl pCC1FOS™ and pCC2FOS™ Vectors for CopyControl Fosmid library production are supplied linearized at the Eco72 I (blunt) site and then dephosphorylated. The vector is ready for cloning end-repaired (blunt-end) genomic DNA of approximately 40 kb.

i_copycontrol_vectorprimer.gif
Figure 2. The CopyControl™ pCC2FOS™ Vector primer cassette. The vector differs from the pCC1FOS Vector by the engineering of a new primer cassette that eliminates wasteful vector-derived sequencing reads and minimizes the potential for priming on the E. coli genome.

i_copycontrol_overviewprocess.gif
Figure 3. Overview of the process for preparing a fosmid library using the CopyControl™ Fosmid Library Production Kits. Once the library has been prepared, individual clones can be cultured in small volume and induced to multiple-copy number for high yields of high-purity DNA for fingerprinting, sequencing, etc., using Epicentre's DirectLysis Fosmid96 kit or FosmidMAX™ DNA Purification Kit.


c_copycontrol_typicalsequencing.gif
Figure 4. Typical sequencing results obtained with the pCC2FOS™ Forward Primer on a pCC2FOS™ clone at 1/48x BigDye™ dilution. Similar results were obtained with the pCC2FOS Reverse Primer (data not shown).


g_copycontrol_fosmidclones.jpg
Figure 5. CopyControl™ Fosmid clones can be induced up to 50 copies per cell to greatly increase DNA yield. Hind III digests of fosmid DNA isolated from uninduced (–) and induced (+) CopyControl clones. Digests contained one-third (8 µl) of the total sample volume and were analyzed by agarose gel electrophoresis. Lane M, Kilobase ladder.

Citations
  1. FEMS Microbiol Lett 284 (2008) 28-34


EpiFOS™ Fosmid Library Production Kit

Applications
  • Preparation of complete and unbiased fosmid libraries that can be maintained at single-copy number.

The EpiFOS™ Fosmid Library Production Kit provides all reagents needed to construct up to 10 complete and unbiased primary fosmid libraries. Fosmid clones constructed in the pEpiFOS-5 Vector cannot be induced to high-copy number, limiting the DNA yields during purification. For this reason, Epicentre researchers prefer the use of the CopyControl™ Fosmid Library Construction Kit in the construction of their genomic libraries.

Primers for end-sequencing pEpiFOS-5 clones are available separately.

Benefits
  • EpiFOS Vectors are supplied Cloning-Ready: linearized, dephosphorylated, purified, and ready for ligation.
  • No need for partial restriction endonuclease digests or pulse-field gel electrophoresis to prepare the genomic DNA for cloning.
  • High-efficiency lambda packaging eliminates background and false positives.
  • Faster and easier than BAC cloning.

pepifos-5.gif
Figure 2. EpiFOS™-5 Fosmid Vector.

pepifos_bac_cloning_horiz.gif
Figure 1. Production of a fosmid library using the EpiFOS™ Fosmid Library Production Kit.



CopyControl™ Fosmid Autoinduction Solution

The CopyControl™ Autoinduction Solutions* are designed to provide a simpler way to induce CopyControl Fosmid and BAC vectors to high-copy number. The Autoinduction Solution, which is added prior to culture inoculation, is designed to cause induction to occur prior to DNA purification without the need for subculturing. The simple, "hands-off" protocol is particularly useful in high-throughput purifications where subculturing is tedious and inefficient.

In addition to the simpler induction protocol, CopyControl Autoinduction Solutions also contain ingredients which enhance cell growth, leading to higher culture densities, and subsequently higher DNA yields than are typically seen using the standard CopyControl Induction Solution.

The two Autoinduction Solutions are cloning-system-specific and not interchangeable, so the proper Autoinduction Solution must be used with each cloning system, as indicated, for optimum results.

g960605bm96.jpg
Figure 1. Uninduced (single-copy)(-) and autoinduced(+) CopyControl™ BACs purified using the BACMAX96™ DNA purification system.When used in conjunction with the BACMAX96 system, autoinduced CopyControl vectors typically yield over 3 µg of DNA using 1.2 ml of starting culture.

*Covered by issued and/or pending patents.



CopyControl™ BAC Cloning-Ready Vectors

Applications
  • Preparation of BAC libraries that can be maintained at single copy number and induced to high-copy number as needed.
CopyControl™ BAC Cloning-Ready vectors are linearized and dephosphorylated cloning ready vectors that contain the E. coli F-factor single-copy origin of replication and inducible 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 Autoinductionor CopyControl Induction Solutions to the culture, switching on the trfA/oriV host cell/vector-borne combination to provide higher yields of BAC DNA. The short induction times of CopyControl BAC clones in the presence of either of the two inductions to high-copy number does not decrease clone stability (based on analysis ofHind 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 is added to the culture medium prior to inoculation, and also increases the cell growth leading to higher yield than standard Induction Solution.

Phage T1-resistant TransforMax™ EPI300™-T1R 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.

Benefits
  • Safely obtain higher DNA yields while maintaining the stability of single-copy-number clones.
  • Constructing BAC libraries is faster and easier.
  • CopyControl pCC1BAC cloning vectors are Cloning-Ready: linearized, dephosphorylated, and highly purified.
  • Cloning-Ready pCC1BAC vectors give >95% white colonies.
  • Estimate the size of the CopyControl BAC clones in as little as 4 hours without culture,Not I digests, or pulse-field gel electrophoresis.
  • CopyControl BACs are ideal for use in high-throughput (96-well) purifications when used with the CopyControl BAC Autoinduction solution.
i_copycontrolbac_protocol.jpg
 Figure 3. The CopyControl™ BAC Cloning Kits significantly reduce the time and labor needed to construct a BAC library compared to standard methods.

cc_bac_fig4a.jpg
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. 

cc_bac_fig4b.jpg
 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.
SYBR® is a registered trademark of Molecular Probes, Inc.



pWEB-TNC™ Cosmid Cloning Kit
pWEB™ Cosmid Cloning Kit

Applications
  • Preparation of complete, unbiased cosmid libraries.
The pWEB-TNC™ and the pWEB™ Cosmid Cloning Kits facilitate rapid and efficient construction of cosmid libraries using pWEB-TNC (Fig. 1) or pWEB (Fig. 2) Cosmid Vectors, respectively. The kits use the strategy of cloning end-repaired, randomly sheared DNA instead of the conventional approach of cloning fragments generated by partial restriction endonuclease digestion. The sheared DNA is end-repaired to generate 5´-phosphorylated blunt ends and size-selected using a low-melting-point agarose gel. The size-selected DNA is then ligated into the supplied linearized and dephosphorylated pWEB-TNC or pWEB Cosmid Vector, packaged using ultra-high efficiency MaxPlax™ Lambda Packaging Extracts (>109pfu/µg for phage lambda) and plated on phage T1-resistant EPI100™-T1R E. coli plating cells, all included in the kit. The result is a complete and unbiased primary cosmid library.

Epicentre also offers kits for fosmid cloning and library production. See related products for more information.

Benefits
  • pWEB cloning vectors are cloning-ready: linearized, dephosphorylated, and highly purified.
  • No need for partial restriction endonuclease digests or pulse-field gel electrophoresis to prepare the genomic DNA for cloning.
  • High-efficiency lambda packaging eliminates background and false positives.

 
i_pweb-tnc_vector.gif
 
i_pweb_vector.gif
 Figure 1. Map and cloning region of the pWEB-TNC™ Vector. TheSma I cloning site is flanked by pairs ofBamH I, EcoR I, and Not I sites to aid in the excision and mapping of insert DNA.  Figure 2. Map and cloning region of the pWEB™ Vector. GenBank accession number AF075573.



 i_pweb_proc.gif
 Figure 3. Outline of the cosmid cloning procedure using the pWEB-TNC™ and the pWEB™ Cosmid Cloning Kits.



MaxPlax™ Lambda Packaging Extracts

Applications
  • Packaging of lambda, cosmid, or fosmid DNA for construction of genomic libraries.
MaxPlax™ Lambda Packaging Extracts offer maximum in vitro packaging efficiencies of cos site-containing methylated or unmethylated DNA. Traditional lambda packaging extracts are derived from two complementary lysogenic E. coli strains, BHB2690 and BHB2688, as described by Hohn.1 MaxPlax Extracts use a restriction-free E. coli K-12 strain, NM759, in place of strain BHB2690.2 The sonicated extracts of NM759 are combined with extracts of BHB2688 to produce MaxPlax Extracts.

Each lot of MaxPlax Extracts is tested and guaranteed to maintain a packaging efficiency of greater than 1 x 109 pfu/µg of Control Ligated Lambda DNA for 1 year from the date of purchase if stored properly. A Certificate of Analysis stating actual packaging efficiency (pfu/µg DNA) is provided with each lot of MaxPlax Extracts.


 Benefits
  • Packaging efficiencies of up to 3 x 109 pfu/µg DNA.
  • MaxPlax Extracts are devoid of all known restriction activities. Packages highly methylated DNA as efficiently as unmethylated DNA.3
  • No premixing of different extract components required before use.
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References
  1. Hohn, E.G. (1979) Methods Enzymol. 68
  2. Gunther, E.G. et al. (1993) Nucleic Acids Res. 21, 3903.
  3. Fiandt, M. (2000) Epicentre Forum 7(3), 14.
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