I have synthesized ssRNA by incorporating T7 promoter in a strand of DNA. After pcr I purified it. Next in vitro-transcription of template was done at 37 deg for 3 hours.
20ul reaction
template: 10 ul(100ng)
rntps(25mM): 0.4ul (.5mM)
ribolock: 5ul
water: 7ul
enzyme(50U/ul): 5ul (1U/ul)
are you sure that it can be done? tgge is designed for separation of fragments with the same size but different sequence composition and it is based on step by step dsDNA opening over the temperature gradient. The whole dsDNA opens and opens more and more and finally it stops in the gel because of conformation. Both strands are "glued" by GC clamp. Im not sure, if tgge can be used for RNA. probably yes for dsRNA and more more more probably no for ssRNA. ssRNA cant gradually melt and therefore there will be no mobility shift. dsRNA can melt, but you will and with 2 separated bands for each RNA.
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