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polyA tailing bacterial RNA

Marta
in Biochemistry and Molecular Biology

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Does anyone do polyAtailing after obtaining mRNA from bacteria? I have some questions:

1) what is the minimal quantity of mRNa for polyAtailing. The protocol epicentre is designed for 1-10ug (alternative protocol) but I wonder that lower concentration are also good, for example 0,3 ug?

2) Does anyone check the shift after polyAtailing on the 1% denaturing gel? I have some problems because I do not see the shift between mRNA and mRNA polyAtailed and I wonder if it may look like that (bacuase reagents of kit are good for sure and mRNa is also good-purified with minelute Qiagen)

 

I will be ver gratefull for answers. I would like to start cDNA synthesis with oligoDT but I am not sure about my last step with polyAtailing. If I do not see the shift, what to do?

I hew new isolations, new kit...

HELP me,please

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