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[slightshiner]

Hi everyone, I stuck in the RNA ligase recently and need your suggestions..

Here is my problems:

I need one 200 bs RNA (accurate sequence). I designed my ligation reaction. 1. 100 bs RNA piece (3-OH donor); 2. 100 bs RNA piece with 5' phosphate; 3. 20 bs DNA complementary template.

First step, annealing two piece RNA (1/1) in STE buffer with the existence of excessive template;
Second step, introducing the T4 RNA ligase 2 (Biolabs) and ligation buffer, 37 oC half an hour.

Then checking with PAGE gel, I found noghitng except the starting two pieces of RNA. I tried it times, but all are same (failed)...


So I tried another reaction, and try to ligate one RNA piece (3-OH donor) and another DNA piece (5'-phosphate) with the existence of corresponding complementary 20 mer DNA template, it failed again....

I re-ordered the ligase from New England Biolabs, and applied the new one in my both types of reaction, failed again....

Too frustrating, I am too tired..

I am sure My 3-OH RNA donor are ok, checked with mass; the 5'-phosphate DNA piece is totally ok, because I succeeded in using it to ligate with another DNA piece (3-OH donor) using T4 DNA ligase....

I cannot figure out what is the problem.....

The STE buffer I used was stocked for long time. Is that a issue caused the failure?
Any suggestion is helpful.....

Thanks.