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mRNA Bioanalyzer


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#1 Hrdinka

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Posted 01 December 2009 - 09:18 AM

Dear forum,

I am really desperate.

I generate mRNA.

I linearise my plasmid and do transcription.

The transcript is 4000 bp long. The plasmid (with the ORF) is 9000 bp long (but I do DNase digestion after transcription).

I get two different patterns on the bioanalyzer (see attachment- first peak marker, second peak about 2000 bp, 3 third peak 4000 bp).

I just can't explain what the 2000 bp peak is. And why I somtimes only get this one.

Is anyone able to help? I would so much appreciate it!!

Thanks Astrid

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  • Bioanalyzer_Forum.jpg


#2 pDNA

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Posted 01 December 2009 - 12:25 PM

could be premature termination due to secondary structure of your mRNA ...or an intrinsic weak terminator.

You linearized your plasmid before RT ...does the cut in the plasmid coincides with the end of your transcript or is there a terminator?

The left image looks weird ...the right looks quite good (sharp peak).

Maybe you can give more information on your transcript (mammalian/prokaryotic cDNA used)?

Regards,
p




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