Product Name: Thermus aquaticus MutS DNA mismatch repair protein
Product Type: Purified recombinant protein
Source: E. coli.
Fusion: 6XHis-MBP at N-terminus
Size: 500 µg
Concentration: 1 mg/ml
Gene Name: Thermus aquaticus MutS DNA mismatch repair protein
Accession #: AAC43637, TAU3311, U33117
GI: 1203807
Purification Methods: FPLC
Purity: >95%
Storage: -20 oC
Storage Buffer: 20 mM Tris-HCl, pH 8.0, 150 mM imidazole, 250 mM NaCl, 1 mM DTT, 50% Glycerol
QC: SDS-PAGE
Applications:
1. Remove mismatch DNA (error correction) from gene synthesis reaction
2. Mutation detection and removal.
Example Protocol
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 oC for 5 min followed by cooling at 0.1oC/s to 25 oC.
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 6XHis-MBP–MutS dimers to 950 nM.
4. Incubate the mixture at room temperature for 10 min, then add an equal volume of amylose resin (NEB) preequilibrated with 1X 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).
Ref:
1. Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Res. 2004 Nov 23;32(20):e162.
2. Correcting errors in synthetic DNA through consensus shuffling. Nucleic Acids Res. 2005 Mar 30;33(6):e55.
3. MutS as a tool for mutation detection. Acta Biochim Pol. 2005;52(3):575-83. Epub 2005 Aug 4.
4. One tube mutation detection using sensitive fluorescent dyeing of MutS protected DNA. Nucleic Acids Res. 2000 Apr 15;28(8):E36.
