I've had the immense joy of attempting to sequence an in vitro
transcribed pol II transcript by doing primer extension in the presence
of ddNTP's. I get plenty of RT run off in controls (rxn's containing
only dNTP's) and only once have I gotten faint ladders. I've tried
increasing the ammount of ddNTP's and even reducing them but to no
avial. Does someone out there have conditions which produce readable
sequence ladders? Or just some general tips in doing these kinds of
things. My RNA is produce by human nuclear extracts in a run off assay
(acutally beaded templates for those in the know) which produce pretty
clean txn's. One usefull thing I have found out is that yRNA, used as a
carrier, is inhibitory to RT when present in excess of 80µg.
I'm useing MMLV to do the primerextension with a labeled primer.
The really fun part about this is I worked out conditions that give a
great sequenceing ladder with a T7 derived transcript but when I use
those conditions with my pol II RNA all I get is run off. HELP!!!!!
I'm going insane and I need this for my commitee meeting in a couple of
Thomas at molbio.uoregon.edu