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much smaller fragments after IVT


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#1 mahrak

mahrak

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Posted 30 December 2012 - 10:52 PM

hey there all


I have been trying to do a round of In vitro transcription using TranscriptAid T7 High Yield Transcription Kit( fermentas):
1- I linearize my plasmid with BamHI and perform phenol/chloroform extraction followed by ethanol precipitation.
2- I set up the IVT reaction acording to manual and let sit at 37centigrade degrees for 2 hrs
3- I treat the reaction with DNase I ( kit supplemented)
4- I perform another round of phenol/chloroform extraction followed by ethanol precipitation to have Purified RNA
5- I check the concentration and purity by spect.
6- I run the produced RNA on gel along with an RNA ladder, unfortunately the size is much, much, much smaller than what I expect; desired RNA should be app. 1500base in lenght, what I observe on gel is 100base.
I already have checked for t7 termination sequences on my plasmid. there are none. so I expect the t7 polymerase to "run off".
HELP!!
HELP!!




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