Editing Muts Activity Assay (section)

== DNA protection assays:&nbsp;DNA binding of MutS using SYBR-Gold MutEx assay ==

1. PCR two fragments<br/>one from 2400 pET30b-hRAD51<br/>another one from 2397 pDNR-Dual-hRAD51<br/>2400-hRAD51-Nde1-F<br/>tagcaacatATGGCAATGCAGATGCAGCTTG

2400-hRAD51-Xho1-R<br/>(GAGTGGGAGATGCCAAAGACctcgag tagcaa)<br/>ttgctactcgagGTCTTTGGCATCTCCCACTC



<br/>catatggcaatgcagatgcagcttgaagcaaatgcagatacttcagtggaagaagaaagc

tttggcccacaacccatttcacggttagagcagtgtggcataaatgccaacgatgtgaag

aaattggaagaagctggattccatactgtggaggctgttgcctatgcgccaaagaaggag

ctaataaatattaagggaattagtgaagccaaagctgataaaattctggctgaggcagct

aaattagttccaatgggtttcaccactgcaactgaattccaccaaaggcggtcagagatc

atacagattactactggctccaaagagcttgacaaactacttcaaggtggaattgagact

ggatctatcacagaaatgtttggagaattccgaactgggaagacccagatctgtcatacg

ctagctgtcacctgccagcttcccattgaccggggtggaggtgaaggaaaggccatgtac

attgacactgagggtacctttaggccagaacggctgctggcagtggctgagaggtatggt

ctctctggcagtgatgtcctggataatgta

gcC tat gcG cga

gcgttcaacacagaccac

cagacccagctcctttatcaagcatcagccatgatggtagaatctaggtatgcactgctt

attgtagacagtgccaccgccctttacagaacagactactcgggtcgaggtgagctttca

gccaggcagatgcacttggccaggtttctgcggatgcttctgcgactcgctgatgagttt

ggtgtagcagtggtaatcactaatcaggtggtagctcaagtggatggagcagcgatgttt

gctgctgatcccaaaaaacctattggaggaaatatcatcgcccatgcatcaacaaccaga

ttgtatctgaggaaaggaagaggggaaaccagaatctgcaaaatctacgactctccctgt

cttcctgaagctgaagctatgttcgccattaatgcagatggagtgggagatgccaaagac

ctcgag

3. Purify DNA fragments



4. in a PCR tube

Add 4 ul of 10x concentrated PCR buffer&nbsp;(100 mM Tris–HCl (pH 8.8 at 25 °C), 500 mM KCl,&nbsp;1.0% Triton X-100, and 18 mM MgCl 2 ),

add&nbsp;water to 40 ul.

5.&nbsp;Heat for 2 min at 90 °C so as to denature DNA.<br/>then 60 oC for 2 min and then to 37 oC for 2 min.

6. Add&nbsp;tenfold molar excess of Taq-Muts

7. Incubate at 60 °C for 5 min to form&nbsp;DNA–MutS complexes,

8. Transfer to 37 °C&nbsp;for 2 min, followed by the addition of 1 ul (5 U) T4&nbsp;DNA polymerase

9. Incubate at 37 °C for further 10 min. Then, add 0.5 ul of 100X&nbsp;SYBR-Gold&nbsp;solution prepared from 10,000 X

10. Monitor green fluorescence under blue light illuminator