Paul.xu at 19:18, 12 August 2016

2016-08-12T19:18:00Z

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

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

2400-hRAD51-Xho1-R (GAGTGGGAGATGCCAAAGACctcgag tagcaa) ttgctactcgagGTCTTTGGCATCTCCCACTC

 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 (100 mM Tris–HCl (pH 8.8 at 25 °C), 500 mM KCl, 1.0% Triton X-100, and 18 mM MgCl 2 ),

add water to 40 ul.

5. Heat for 2 min at 90 °C so as to denature DNA. then 60 oC for 2 min and then to 37 oC for 2 min.

6. Add tenfold molar excess of Taq-Muts

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

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

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

10. Monitor green fluorescence under blue light illuminator

== 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 (100 mM Tris–HCl (pH 8.8 at 25 °C), 500 mM KCl, 1.0% Triton X-100, and 18 mM MgCl 2 ),

add water to 40 ul.

5. Heat for 2 min at 90 °C so as to denature DNA. then 60 oC for 2 min and then to 37 oC for 2 min.

6. ?? 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 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.

== DNA retardation assay ==

Add 4 ul of 10x concentrated buffer (50 mM MgCl 2; 100 mM Tris–HCl, pH 9.0, 500 mM KCl, and 1% Triton X-100),

Add 4 ul ATP (10 mM). 

Add DNA fragment

Add water to 40 ul 

eat to 92~95 °C for 5 min

Cool to 62 °C for 10 min (or reannealing 670C 1 hr), and to 22 °C for 5 min to form heteroduplexes.

Add  6xHis Tth MutS

Incubate  at 60 °C for 10 min.

Run gel at room temperature, in 5% polyacrylamide gel, in 89 mM Tris–borate buffer, pH 8.3, with MgCl2 addition to 1 mM at 10 V/cm for 2 h, followed by staining with SYBR-Gold

== Colorimetric Assay ==

Preparation of Tested DNA Fragments The DNA fragments were prepared from synthetic oligonucleotides. The biotinylated oligonucleotide T49, BIOT-ACAGATCCACTGTGCGACGAGCTGTGCCGCACGGTGATCGCAGCCGCTG was mixed with an equimolar amount of fully complementary A49, CAGCGGCTGCGATCACCGTGCGGCACAGCTCGTCGCACAGTGGATCTGT or almost fully complementary G49 CAG CGGCTGCGATCACCGTGCGGCGCAGCTCGTCGCACAGTGGATCTGT in order to obtain fully complementary or mismatched DNA fragments, respectively.

*T49 ACAGATCCACTGTGCGACGAGCTG T GCCGCACGGTGATCGCAGCCGCTG
*A49 ACAGATCCACTGTGCGACGAGCTG T GCCGCACGGTGATCGCAGCCGCTG (rev comp)
*G49 ACAGATCCACTGTGCGACGAGCTG C GCCGCACGGTGATCGCAGCCGCTG (rev comp)

To prepare oligonucleotide duplexes, the mixtures of HPLC purified oligonucleotides were suspended in a buffer containing 10 mMTris–HCl (pH 8.8/25 oC), 50 mMKCl, 0.08 %(v/v) Nonidet P40 and 5 mMMgCl2 in a final volume of 100 ul to a final concentration of 10 uM, and subjected to heating and cooling (92 oC/120 s, 65 oC/120 s, 25 oC/120 s).

The microplate wells (Pierce Nickel Coated Plates, (Thermoscientific, cat. no. 15442, lot #NH174556) were unblocked by washing with five 200 ul portions of washing/ binding buffer WB (1 X PBS buffer (Sigma, cat. no 79383, lot. BCBF0586) supplemented with 1 % of Tween- 20 (Cat. No P7949, lot #SZ2B2070V) and 5 mM MgCl2. One ug of his-tagged MutS protein was loaded per one microplate well in 100 ug of the same buffer. After 15 min of incubation, the well was emptied, following washing with five 200 ul portions of WB buffer and immediate loading of the tested biotinylated DNA (10 pmol) in 100 ul of WB buffer. After 10 min of incubation, the excess of unbound DNA was removed by washing with five 200 ul  portions of WB buffer. DNA binding by the immobilized protein was detected with horseradish peroxidase conjugated to avidin (Extravidin, Sigma, cat. no. E2886, lot 098K4751). The well was loaded with 100 ul  of Extravidin 1000 times diluted in WB buffer and after 10 min of incubation washed with five 200 ul  portions of WB buffer, following immediate loading of 100 ul  of TMB (3,30,5,50- Tetramethylbenzidine) chromogenic substrate solution (Sigma, cat. no T0440, lot 051M1850). The colour reaction was developed for 15 min, and the absorbance was measured with an ELISA reader (MultiSkan FC, ThermoScientific, cat. No. #51190000) at 620 nm. All steps of the protocol were carried out at room temperature.
[[Category:Protocols]]

Muts Activity Assay - Revision history

Paul.xu at 19:18, 12 August 2016