Sense or antisense? How can I tell directionality of insert? - Trying to make an antisense riboprobe via in vitro transcription with DIG-UTP fo (Jun/28/2006 )
I have a clone that was mailed to me from another lab but it didn't say which restriction enzyme/polymerase combination to use to make antisense. I had the plasmid sequenced, and when I BLASTed the insert against genbank, using the sequence obtained from sequencing with T7, the BLAST hits were all strand=+/+, which i understand to be "sense", and sequencing from T3, the hits were strand=+/-, which i understand to be the reverse complement of the gene. So which polymerase should I use to synthesize antisense RNA probe, T7 or T3? In other words, if I transcribe from the +/+("sense") DNA strand, does that give me antisense RNA or sense message?
Thanks in advance for any advice!
The antisense DNA is transcribed in the sense mRNA, therefore you should use the T3 polmerase to obtain the sense mRNA and the T7 to sintesize the antisense mRNA. This link can clarify better: