I have cloned the fragment of interest me cDNA to pCS2+ vector to perform transcription in vitro. I have checked by sequencing the sides for SP6 (sense probe) and T7 (antisense probe) polymerases. They are in the vector. I have done transcription and precipitation with LiCl (2 h) and I got only sense probe. I have repeated procedure for antisense probe two times more with the same results. I have no idea what could be the reason of this problem. I will be grateful for any suggestions.
Thank you in advance,
Lidia
Is your probe sequence the same as the sequence that you wish to probe?
I have already sequenced vector with insert (cDNA). It looks fine. I hope transcription with SP6 gave me sense oriented probe. Now I am going to prepare PCR with reverse primer containing promoter for the T7 RNA polymerase.
OK, you might want to take a look at this: http://www.protocol-online.org/biology-forums/posts/31067.html
It can be confusing - you want to synthesize from the T7 side.
The vector pCS+ does not appear to have a T7 promoter site, it has rather a sense SP6 and antisense T3 site, which is why probably you are only getting the sense probe but no antisense one.. An easy and clean approach is just design a primer with T7 on 5' of the reverse primer, get a PCR product with T7 promoter on the antisense strand and use this product as template for in-vitro transcription
See if that helps
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