 | | | | ET SSB | | | Developed and produced by BioHelix Corp., a NEB-affiliated company. | |
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Prices are in US dollars and valid only for US orders.
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- Can withstand extremely high temperature conditions
- Ideal for nucleic acid amplification and sequencing
- Isolated from a recombinant source
Description:
ET SSB (Extreme Thermostable Single-Stranded DNA Binding Protein) is a single-stranded DNA binding protein isolated from a hyperthermophilic microorganism, which remains fully active after incubation at 95°C for 60 minutes. Due to the extreme thermostability, ET SSB can be used in applications that require extremely high temperature conditions, such as nucleic acid amplification and sequencing.
Figure 1: improved multiplex PCR by ET SSB. By adding ET SSB, specific amplification was accomplished in PCR using one to five primer pairs in incurring order (lanes 1-5). Lane 5C shows a PCR reaction using 5 primer pairs, with no ET SSB included (control reaction). Lane M: 100 bp DNA Ladder.
Figure 2: Multiplex HDA was improved by ET SSB. The HDA reactions were prepared with either single or two sets of primers to amplify the invE (A) and/or invA (B) from Salmonella typhimurium genomic DNA. Lane M: Low Molecular DNA Ladder; Lane A: invE gene; Lane B: invA gene; Lane AB: invE and invA genes with no ET SSB present; Lane AB**: invE and invA genes with ET SSB in the HDA reaction.
** the reaction contained ET SSB.
Source:
An E. coli strain that carries the cloned ssb gene from a hyperthermophilic organism.
Applications:- Improve the processivity of DNA polymerase (1)
- Stabilization and marking of ssDNA structure (2)
- Increase the yield and specificity of PCR reactions (3-7)
- Increase the yield and processivity of RT during RT-PCR (8-9)
- Improve DNA sequencing through regions with strong secondary structure (6)
- Enhance the RecA activity for ssDNA binding and strand transfer (10,11)
Reaction & Storage Conditions
Unit Definition:
Sold by mass of pure protein as determined by OD280.
Concentration:
1 mg/ml
Storage Conditions:
20 mM Tris-HCl
200 mM NaCl
0.5 mM DTT
1 mM EDTA
50% Glycerol
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- ET SSB is active in any polymerase buffer. Add 200 ng of ET SSB per 50 µl reaction.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
ET SSB is purified free of contaminating endonucleases and exonucleases. Each lot is tested for ssDNA binding activity and is visually determined to be > 95% pure on an SDS-polyacrylamide gel.
Exonuclease Activity:
Incubation of 20 µg ET SSB for 4 hours at 65°C in 50 µl reaction buffer containing 50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate and 1 mM dithiothreitol (pH 7.9 at 25°C), with a mixture of single and double-stranded [3H] E. coli DNA (200,000 cpm/µg) released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of 7 µg ET SSB for 4 hours at 65°C in 50 µl reaction buffer containing 50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate and 1 mM dithiothreitol (pH 7.9 at 25°C), with 1 µg ΦX174 RF I DNA gave < 20% conversion to RF II.
Nuclease Activity:
Incubation of 20 µg ET SSB for 16 hours at 65°C in 50 µl of reaction buffer containing 50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate and 1 mM dithiothreitol (pH 7.9 at 25°C), with 1 µg λ DNA yielded a clear and sharp band on an agarose gel.
References
- Myers, T.W. and Romano, L.J. (1988) J. Biol. Chem., 263, 17006-17015.
- Reddy, M.S. (2000) Biochemistry, 39, 14250-14262.
- West, S.C., Cassuto, E. and Howard-Flanders, P. (1982) Mol. Gen. Genet., 186, 333-338.
- Goldmeyer, J., Kong, H., and Tang, W. (2007) J. Mol. Diagnostics, 9, 639-644.
- Delius, H., Mantell, N.J. and Alberts, B. (1972) J. Mol. Biol., 67, 341-350.
- Schwarz, K., Hansen-Hagge, T. and Bartram, C. (1990) Nucl. Acids Res., 18, 1079.
- Chou, Q. (1992) Nucl. Acids Res., 20, 4371.
- Oshima, R.G. (1992) Biotechniques, 13, 188.
- Rapley, R. (1994) Mol. Biotechnol., 2, 295-298.
- Olszewski, M. et al. (2005) Mol. Cell Probes, 19, 203-205.
- Baugh, L.R. et al. (2001) Nucl. Acids Res., 29, e29.
- Villalva, C. et al. (2001) Biotechniques, 31, 81-83, 86.
Legal
Licenses/Patents/Disclaimers:
Some applications in which this product can be used may be covered by patents issued and applicable in the United States and certain other countries. Because purchase of this product does not include a licenses to perform any patented application, users of this product may be required to obtain a patent license depending upon the particular application in which the product is used.
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