cy5 labelt RNA impossible to stain with sybrgreen II ? - (Jul/12/2007 )
i am working on a functional gene oligonucleotid microarray.
usually i prepare my targets in this order: PCR (+ t3 promotorsite), IVT invitro transcription (25% cy5 UTP's) follod by a fragmentation and a qualitie control on a agarose gel.
and surprisingly it's not possible to stain the "cy5" labelt RNA.
wenn i use unlabeled RNA, sybrgreen II works realy good!
by "cy3" labeled RNA sybrgreen II works not good but enough.
and with cy5 labeled RNA ----- nothing.
i know that there is enough RNA on the gel but i don't understand it.
i also tried EtbR and sybrgreen I.
if someone can help me, i would be deeply greatful.
Could it be the effect of atmospheric ozone? If the ozone level is high, the Cy5 signal might be bleached.
Have seen the same here with EtBr. The more Cy5 is incorperated the less the etbr intercalates with the nucleotides