In vitro transcription: why double-banded result? - (Oct/21/2008 )
I am using T7 polymerase to in vitro trancribe linerized (with SpeI) TOPO 2.1 plasmid with my insert (1.5kb). After SpeI digest, I check to make sure I have only a single band of linearized plasmid, then I purify it from the gel, so nothing from the digest will interfere with the transcription. Then I use Takara T7 polymerase to transcribe it at 42°C for 1.5 hours. I clean it up with Qiagen RNeasy and then visualize it on an acrylamide gel. Instead of getting a single product from my single linearized plasmid, I get two nice bands, both near the expected size.
Does anyone have any ideas why this could be happening?
Thanks,
Karen
It also happened to me and then i looked at the vector and realized that it contained two active T7 promoters 
facing each other with my insert between them.
facing each other with my insert between them.Yeh, I'm using Invitrogen Topo 2.1 and it only has one T7 promoter, but thanks for the tip!
Actually, I think I (with the help of an Invitrogen tech and my labmates) figured out the problem. The extra band on the gel is 250bp longer than the correct product. Even though SpeI cuts with a blunt end, I didn't dephosphorylate. So some of the plasmid may have re-connected. Therefore, T7 may have had the freedom to move past the SpeI cut site and reach some other stop sequence 250 bp later. So, I could dephosphorylate after SpeI, or I could do a double digestion with an enzyme that cuts the rest of the plasmid up while leaving T7 to Spe I (including my insert) untouched. But, I think I'll just try what I should've done in the first place: amplify the plasmid with M13f-M13r (on either side of my insert) and gel purifying so T7 can bind to a short PCR product. Since I don't need any parts of the plasmid besides the insert and the T7 promoter, this should make things less complicated. If someone thinks this is a bad idea, please let me know why. Otherwise, I hope someone else can use this info!
that is exactly the same thing that i did and it worked. i just had to pool some pcr reactions to get enough material.