A siRNA is replaced in the sense and anti sense regions of an Mirna without disturbing it's backbone and is placed in a synthetic intronic region. I have the intronic region with a poly linker sequence obtained commercially and tat was cloned into pcdna3.1. I actually do anneal the oligos at 95 degC with annealing buffer followed by the Kinase treatment (very low concentration ) and I go for overnight ligation. This will replace the poly linker with amirna. Yet, I get negative clones. I don't elute my vector so tat I can have a trial in getting positive clones if any after many trials of elution. I even tried to treat the oligos separately with Kinase and then go for annealing which gives higher concentration of annealed oligos with purity more than 2.3. I use the ratio as 1:3 for ligation (V:I). After so many trials, I m unable to get positive clones. Any suggestions would be helpful and appreciable towards fixing it. Thank you all in advance.
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