IMB205

a：Concentration for test : 0.01、0.1、0.5 (for regular nucleases ~ nM level )、1、2、5( for low activity nucleases ~ mM level)…，擇一活性較佳濃度

    (1 μM以下為較佳活性濃度)

 b：Amount of Substrate: for plasmid - 100ng~200ng ; For Oligo DNA substrate -25~300ng at the reaction volume around 10ul擇一適當濃度.

 c：Optimized concentration of salt : NaCl 25~1000mM，擇一活性較佳濃度

 d：Optimized concentration of metal ion : 依蛋白需要適量加入

       Final concentration for respective ion:

Mg²⁺(~1mM)

Zn²⁺(~1mM) (zinc~0.2 mM in E.coli)

Ca²⁺(calcium ~100nM calcium in vertebrates /~0.2 mM in E.coli)

Outten CE, O’Halloran TV (2001) Science 292:2488–2492

37℃ incubation for 1 hrs (反應所需時間依實驗需求調整)

1.  加入Incubate with 1 μl protease K，37℃ 0.5 hrs

2.  跑Apply incubated samples for electrophoresis analsis (Native Gel、TAE、TBE或TBE- UREA gel)分析實驗結果

   Most of the cases, 6X DNA loading dye for native Gel, 2X UREA loading dye for Urea denature Gel.

Tips：

       1.      若以20% TBE gel分析，EtBr stain染膠30sec~1min，退染distain>20min

       2.      anneal complementary pairs of oligonucleotides

       5x annealing buffer：50mM Tris-HCl, 100mM NaCl, 5mM EDTA ,pH8

           加入適量濃度complementary oligonucleotides ( 1:1 molar ratio)

              加入1 x annealing buffer

       95℃ heat for 5 minutes，降至室溫(約take about 50min to reach room tempearture )

       store at -20℃

3.      High temperature among electrophoresis may denature ds nucleotide substrate

4.      Endogenous bound metal ions in purified recombinant protein may be sufficient for digesting affair. Such an issue should be considered for experiment layout.