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| Download: | | Manual|MSDS PDF | |
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Description:
The NEBNext® Fill-in and ssDNA Isolation Module has been optimized to fill in adapter sequence from a nick generated by ligation of adaptors lacking a 5´-phosphate to a DNA template coupled with ssDNA isolation. Bst DNA Polymerase, Large Fragment, recognizes the nick resulting from the ligation of unphosphorylated adapters, displaces the nicked strand and extends the 3´ end of the DNA template and fills in the adapter sequence to generate full length dsDNA. Hydrophilic Streptavidin Magnetic Beads and buffers are provided to allow binding of dsDNA fragments bearing one biotinylated adapter prior to adapter fill-in and elution of the full length, unbiotinylated ssDNA strands after adapter fill-in.
The NEBNext Fill-in and ssDNA Isolation Module is provided as a master mix to maximize efficiency and convenience in DNA sample preparation workflows (1,2,3). The NEBNext Fill-in and ssDNA Isolation Module has been validated by sequencing with the Roche 454 GS FLX Titanium in conjunction with the NEBNext End Repar Module, and the NEBNext Quick Ligation Module.
The Module Includes:
The volumes provided are sufficient for preparation of up to 20 reactions (NEB #E6071S) and 100 reactions (NEB #E6071L).
Box 1: Store at -20°C
Bst DNA Polymerase, Large Fragment
#E6076A: 0.06 ml
#E6076AA: 0.3 ml
Bst DNA Polymerase, Large Fragment is the portion of the Bacillus stearothermophilus DNA Polymerase protein that contains the 5´→ 3´ polymerase activity, but lacks 5´→ 3´ exonuclease activity.
Source: Bst Polymerase, Large Fragment is prepared from an E. coli strain containing a genetic fusion of the Bacillus stearothermophilus DNA Polymerase gene, lacking the 5´→ 3´ exonuclease domain, and the gene coding for E. coli maltose binding protein (MBP). The fusion protein is purified to near homogeneity and the MBP portion of the fusion is cleaved off in vitro. The remaining polymerase is purified free of MBP (1).
Supplied in: 50 mM KCl, 10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% Triton X-100 and 50% glycerol.
NEBNext Adaptor Fill-in Reaction Buffer (10X)
#E6077A: 0.01 ml
#E6077AA: 0.5 ml
New England Biolabs supplies a unique 10X reaction buffer for Bst DNA Polymerase, Large Fragment
1X NEBNext Adapter Fill-in Reaction Buffer:
20 mM Tris-HCl
10 mM (NH4)2SO4
10 mM KCl
2.0 mM MgSO4
0.1% Triton X-100
0.4 mM dATP
0.4 mM dCTP
0.4 mM dGTP
0.4 mM dTTP
pH 8.8 @ 25°C
Box 2: Store at 4°C
Hydrophilic Streptavidin Magnetic Beads (4 mg/ml)
#E6072A: 1 ml
#E6072AA: 5 ml
Hydrophilic Streptavidin Magnetic Beads are 2 μm supermagnetic particles covalently coupled to a highly pure form of streptavidin. The beads can be used to capture biotin labeled DNA.
Supplied in: 4 mg/ml suspension in phosphate buffer (PBS) (pH 7.4) containing 0.1% BSA and 0.02% NaN3.
Support Matrix: 2 μm non-porous magnetic microparticle.
Binding Capacity: The beads will bind greater than 800 pmol of free biotin per mg and greater than 400 pmol of single-stranded 20 bp biotinylated oligonucleotide per mg.
NEBNext Bead Binding Buffer (2X)
#E6074A: 4.5 ml
#E6074AA: 22.5 ml
NEBNext Bead Binding Buffer has been optimized for binding biotin-labeled DNA to Magnetic Streptavidin Beads.
1X NEBNext Bead Binding Buffer:
5 mM Tris-HCl
0.5 mM EDTA
1 M NaCl
pH 7.5 @ 25°C
NEBNext Bead Wash Buffer (1X)
#E6073A: 8 ml
#E6073AA: 40 ml
NEBNext Bead Wash Buffer has been optimized to remove non-specific binding to Magnetic Streptavidin Beads.
1X NEBNext Bead Wash Buffer:
5 mM Tris-HCl
0.5 mM EDTA
1 M NaCl
pH 7.5 @ 25°C
Applications:
- DNA sample preparation
- Adapter fill-in and ssDNA isolation of 1–5 μg fragmented DNA adapter ligand
- Efficient – Enables capture of biotinylated dsDNA or Streptavidin beads. Removes nicks generated by ligation of unphosphorylated primers on 1–5 μg DNA, and enables isolation of full length ssDNA from beads.
- Convenient – Reactions are provided in master mix format to reduce steps during DNA sample prep workflows
- Automation Friendly
Protocols
Protocols for NEBNext® Fill-in and ssDNA Isolation Module
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Control Assays:
Please refer to the Instruction Manual for details.
References
- Maricic, T. and S. Paabo (2009) Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands. Biotechniques, 46, 51-52, 54-57.
- Straus, D. and F.M. Ausubel (1990) Genomic subtraction for cloning DNA corresponding to deletion mutations. Proc. Natl. Acad. Sci. USA, 87, 1889-1893.
- Zhou, X. and D.T. Wong (2007) Single nucleotide polymorphism mapping array assay. Methods Mol. Biol, 396, 295-314.
Companion Products
NEBNext® DNA Sample Prep Master Mix Set 2
NEBNext® DNA Sample Prep Reagent Set 2
NEBNext® End Repair Module
NEBNext® Quick Ligation Module
NEBNext™ dsDNA Fragmentase™