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polyA tailing bacterial mRNA


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#1 Martha

Martha

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Posted 03 May 2011 - 03:53 AM

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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