Editing Muts Activity Assay (section)

== PCR assay ==

Primers T4-RNA-lig-qPCR-F CAAGATATTATGGAAACCTCAGC T4-RNA-lig-qPCR-R CATAAGTCACATCACCGCTTTC



1. Amplify PCR product from 1931 pT7A3-T4-RNA-Lig2 or 123 pT7A3-MBP-(3C)-T4-RNA-Lig2

CAAGATATTATGGAAACCTCAGCGGTTGTATCTTATCAAGTCTTTGGCGAATTCGCTGGACCTGGCATTCAGAAGAATGTTGATTAT TGT GATAAAGATTTTTATGTATTTGACATTATTGTTACTACAGAAAGCGGTGATGTGACTTATG

2. Amplify PCR product from 1127 pEC-T4RNA-Lig2K227Q CAAGATATTATGGAAACCTCAGCGGTTGTATCTTATCAAGTCTTTGGCGAATTCGCTGGACCTGGCATTCAGAAGAATGTTGATTAT GGT GATAAAGATTTTTATGTATTTGACATTATTGTTACTACAGAAAGCGGTGATGTGACTTATG

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.&nbsp;?? ul complex to 1 ug Tth-Muts bind to Ni beads (agarose or magnetic beads).

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

8. Wash beads 3 times with 200 ul TE buffer.

9. Elute DNA using 20 ul of 0.1 N acetic acid. Spin, transfer solution to a new tube (recover beads for reuse)

10. Use 1 ul of eluate for PCR using this pair of primers:

T4-RNA-lig-qPCR-F CAAGATATTATGGAAACCTCAGC T4-RNA-lig-qPCR-R CATAAGTCACATCACCGCTTTC

or do real time PCR using SYBR Green

11. Run on 1% agarose gel.