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Fast-LinkTM, GELaseTM, End-ITTM

DNA Ligation Kit, DNA Repair Kit

Fast-Link™ DNA Ligation Kit

Applications
  • Blunt-end and TA cloning of PCR products.
  • Ligation of next-gen sequencing adaptors to blunt-ended DNA.
  • Genomic DNA cloning and subcloning.
  • BAC/fosmid library construction.
  • cDNA cloning.
  • Linker ligation.
The Fast-Link™ DNA Ligation Kit uses a high-quality ligase (Fast-Link T4 DNA Ligase), that was cloned at Epicentre and formulated to provide extremely rapid, high-efficiency DNA ligation. Cohesive-end ligations can be performed in 5 minutes at room temperature. In contrast to other ligases, it is not necessary to desalt Fast-Link ligation reactions prior to transformation of electrocompetent or chemically competent cells. The Fast-Link Kit can be used for routine and high-throughput DNA cloning.

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Table 1. Fast-Link™ representative results.


Benefits
  • Cohesive-end ligations in 5 minutes at room temperature (Fig. 1).
  • Blunt-end ligations in 15 minutes at room temperature (Fig. 2).
  • Ligation of PCR product with A-overhangs in 1 hour at 16°C.
  • High ligation efficiency (Table 1).
  • Desalting of ligation products is not needed prior to transformation of electrocompetent or chemically competent cells.
  • BAC ligation with inserts >100 kb can be performed in 4 hours, instead of overnight reactions as with other ligases.

g_fastlink_timecourse.jpg
Figure 1. Time course for cohesive end ligation using the Fast-Link™ Kit.Lambda Hind III markers were ligated using 2 units of Fast-Link DNA Ligase (Lanes 2-6). Lane M, Kilobase ladder; lane 1, no enzyme.

g_fastlink_bluntendtimecourse.jpg
Figure 2. Time course for blunt-end ligation using the Fast-Link™ Kit.pUC 19 DNA digested with Pvu II was ligated using 2 units of Fast-Link DNA Ligase (lanes 1-6). Lanes M, Kilobase ladder.



GELase™ Agarose Gel-Digesting Preparation

Applications
  • Recovery of nucleic acids from LMP agarose gels for use in: restriction mapping; cloning; labeling; DNA sequencing;1 transformation of YAC,2,3 BAC,4 cosmid, fosmid, and plasmid vectors; microinjection;2 and amplification.
GELase™ Agarose Gel-Digesting Preparation contains a unique β-agarose digesting enzyme developed at Epicentre for simple, quantitative recovery of intact DNA and RNA from low-melting point (LMP) agarose gels following electrophoresis in TAE, TBE, MOPS, or phosphate buffers. The gel may be digested directly in the electrophoresis buffers or GELase Buffer may be added to, or exchanged with, those buffers for higher activity. GELase Preparation digests the carbohydrate backbone of molten agarose, releasing small, soluble oligosaccharides. The nucleic acid can be used in the digested gel solution or precipitated using ammonium acetate/ethanol. The gel digestion products are alcohol-soluble.

Benefits
  • Recoveries of DNA or RNA consistently approach 100% (Fig. 1).
  • Purify even megabase DNA (Fig. 1) or RNA that is intact and biologically active.1,5,6
  • Simple, flexible protocol with minimal hands-on time.
  • A typical 200-mg gel slice in TAE buffer can be digested in less than 10 minutes using only 3 units of GELase Preparation (Table 1).
  • More active than other gel-digesting enzymes.
  • More economical than spin columns or other gel-digesting methods.
  • Available in two convenient concentrations: 1 U/µl for standard reactions and 0.2 U/µl for greater economy in digesting multiple small gel samples or extending digestion times.

Unit Definition:
One unit of GELase Preparation digests 600 mg (~600 µl) of molten 1% LMP agarose gel in GELase Buffer in 1 hour at 45°C.


Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 0.1 M NaCl, 0.1% Triton X-100, and 1 mM DTT.

Quality Control: Each lot of GELase Preparation is free of detectable DNA exonuclease, endonuclease, and RNase activities.

Notes:
  1. One unit of GELase Preparation equals 3 or more units of most other gel-digesting enzymes.
  2. We recommend adding ammonium acetate for nucleic acid precipitation because GELase Preparation and most other proteins are not precipitated by ethanol in the presence of ammonium acetate. Also, the solubilities of the oligosaccharide digestion products are higher in ethanol in the presence of ammonium acetate. For your convenience, a ready-to-use 5 M Ammonium Acetate Solution is available.

References
  1. Kirkpatrick, H.A. et al. (1997) Epicentre Forum 4(3), 11.
  2. Sasaki, H. and Hogan, B.L. (1994) Cell 76, 103.
  3. Schedl, A. et al. (1993) Nature 362, 258.
  4. Woo, S.S. et al. (1994) Nucleic Acids Res. 22, 4922.
  5. Steck, T.R. (1994) BioTechniques 17, 676.
  6. Chen, L. et al. (1994) BioTechniques 16, 228.

g_gelase_recoverydna_1.jpg
Figure 1. Recovery of any size DNA approaches 100% using GELase™ Gel-Digesting Preparation. In Experiment 1, DNAs from 63 bp to 7.9 kb were separated in a 1.5% LMP-agarose gel (A1), purified using GELase Preparation, and analyzed on a new, 1.5% agarose gel (B1) stained with ethidium bromide. In Experiment 2, high-molecular-weight soybean DNA was separated on a 1% LMP-agarose gel by pulsed-field electrophoresis (A2). The 2.2 Mb DNA band was purified using GELase Preparation, and analyzed on a new 1% LMP-agarose gel stained with ethidium bromide (B2). (Experiment 2 results courtesy of L. Chen and A. Atherly, Dept. of Zoology & Genetics, Iowa State Univ., Ames, IA.)

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Table 1. Time required to digest 200 mg of 1% LMP agarose in TAE buffer. 
i_gelase_protocol.gif
 Figure 2. GELase protocols are simple and flexible, providing highest activity or maximum speed.


End-It™ DNA End-Repair Kit

Applications
  • Repair sheared, nebulized, or restriction enzyme-digested genomic DNA for: preparation of templates for next-gen sequencing; or shotgun library preparation.
The End-It™ DNA End-Repair Kit converts DNA containing damaged or incompatible 5´- and/or 3´-protruding ends to 5´-phosphorylated, blunt-ended DNA. The end-repaired DNA can be rapidly ligated into a cloning vector or to next-gen sequencing adaptors, using the Fast-Link™ DNA Ligation Kit.
The End-It DNA End-Repair Kit contains reagents for 20 or 50 end-repair reactions (repair of up to 100 µg or 250 µg of genomic DNA, respectively).

Benefits
  • The repaired DNA is blunt-ended and 5´-phosphorylated for immediate blunt-end ligation.
  • The high specific activity of the End-Repair Enzyme Mix provides complete conversion of protruding ends to 5´-phosphorylated, blunt-ended DNA.

i_endit_dnafragments_1.gif
Figure 1. DNA fragments containing any type of ends are rapidly and efficiently converted to 5´-phosphorylated, blunt-ended DNA using the End-It™ DNA End-Repair Kit. The end-repaired DNA is ready for blunt-end ligation using, for example Epicentre's Fast-Link™ DNA Ligation Kit.

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