Recombinant Thermus Aquaticus

MutS, Thermus Aquaticus DNA Mismatch Repair Protein, DNA mismatch repair protein MutS.

The MutS DNA Mismatch Protein Identifies heteroduplex DNAs comprising mis-paired or unpaired bases. The MutS DNA Mismatch Protein binds in vitro to heteroduplex DNAs comprising mis-paired or unpaired bases over a varied temperature range from 4-70 °C and obtains a thermostable ATPase activity. The MutS DNA Mismatch Protein is active at temperature between 0 to 75°C. Since The MutS DNA Mismatch Protein competently binds to 1-4 bases deletion (or insertion) and mismatch base pairs of GT, CT and AG, it is suitable for sensing these mutations. Mutations can be distinguished in polyacrylamide gels or on a solid phase such as Ni agarose or beads or magnetic Ni-NTA particles.

DNA Mismatch Repair Protein MutS Thermus Aquaticus Recombinant produced in E.coli is a single, non-glycosylated polypeptide chain containing 829 amino acids and having a molecular mass of 92.8kDa.
The Thermus Aquaticus is fused to a 6 amino acid His-Tag at C-terminus and purified by proprietary chromatographic techniques.

Escherichia Coli.

Sterile filtered colorless solution.

The MutS protein is supplied in 20mM Tris-HCl, pH-8, 250mM NaCl, 0.1mM EDTA, 1mM DTT and 50% Glycerol.

Store at 4°C if entire vial will be used within 2-4 weeks. Store, frozen at -20°C for longer periods of time. For long term storage it is recommended to add a carrier protein (0.1% HSA or BSA). Avoid multiple freeze-thaw cycles.

100mM KCl, 50mM Tris-HCl, pH-8.5, 20mM MgCl₂, 0.1mM EDTA, 1mM DTT and 2% Glycerol, 65 °C.

Greater than 95.0% as determined by SDS-PAGE.

MEGMLKGEGPGPLPPLLQQYVELRDQYPDYLLLFQVGDFYECFGEDAERLARALG

LVLTHKTSKDFTTPMAGIPLRAFEAYAERLLKMGFRLAVADQVEPAEEAEGLVRREV

TQLLTPGTLLQESLLPREANYLAAIATGDGWGLAFLDVSTGEFKGTVLKSKSALYDELF

RHRPAEVLLAPELLENGAFLDEFRKRFPVMLSEAPFEPEGEGPLALRRARGALLAYAQ

RTQGGALSLQPFRFYDPGAFMRLPEATLRALEVFEPLRGQDTLFSVLDETRTAPGRRL

LQSWLRHPLLDRGPLEARLDRVEGFVREGALREGVRRLLYRLADLERLATRLELGRASP

KDLGALRRSLQILPELRALLGEEVGLPDLSPLKEELEAALVEDPPLKVSEGGLIREGYDPD

LDALRAAHREGVAYFLELEERERERTGIPTLKVGYNAVFGYYLEVTRPYYERVPKEYRPV

QTLKDRQRYTLPEMKEKEREVYRLEALIRRREEEVFLEVRERAKRQAEALREAARILAEL

DVYAALAEVAVRYGYVRPRFGDRLQIRAGRHPVVERRTEFVPNDLEMAHELVLITGPN

MAGKSTFLRQTALIALLAQVGSFVPAEEAHLPLFDGIYTRIGASDDLAGGKSTFMVEM

EEVALILKEATENSLVLLDEVGRGTSSLDGVAIATAVAEALHERRAYTLFATHYFELTAL

GLPRLKNLHVAAREEAGGLVFYHQVLPGPASKSYGVEVAAMAGLPKEVVARARALLQ

1. After first round PCR, purify PCR fragments using Qiagen QIAquick PCR purification kit with elution in dH₂O.

 2. Dilute PCR product to 250 ng/µl in 10 mM Tris–HCl, pH 7.8, 50 mM NaCl and heat to 95 °C for 5 min followed by cooling at 0.1°C /s to 25 °C.

 3. Add binding buffer (20 mM Tris–HCl, pH 7.8, 10 mM NaCl, 5 mM MgCl₂, 1 mM DTT and 5% glycerol) to annealed PCR product, adjust DNA concentration to 11.5 ng/µl, add MutS dimers to 950 nM.

4. Incubate the mixture at room temperature for 10 min, then add an equal volume of Ni-NTA beads pre-equilibrated with 1×binding buffer, and incubate for 30 min at room temperature.

5. Gently spin down beads and save supernatant for subsequent processing (second round PCR, cloning etc).

Recommended Applications:

1. Remove mismatch DNA (error correction) from gene synthesis reaction

2. Mutation detection and removal

3. Rapid isothermal SNP detection

SDS

Prospec's products are furnished for LABORATORY RESEARCH USE ONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.