I know that to design a probe for an in situ you need to design an antisense probe that is complimentary to the mRNA of interest.
Then, my problem comes when I try to understand how everything is put together...
We are using TOPO PCRII vectors and we sequenced our fragment with the SP6 primer...
When I BLAST my results, logically, I obtain that some of them follow the pattern:
a) M13R-SP6-→→→→→→→→→-T7-M13F
And some others follow the pattern:
b) M13R-SP6-←←←←←←←←←-T7-M13F
(The arrows indicate the transcription direction of my gene of interest).
My assumption is that if I want to make an antisense probe in the case a) I need to choose the T7 polymerase, and for the case b) I need to chose the SP6 polymerase.
My theory behind that if in case a) we used the SP6pol, it would use the non-coding strand as a template and I would end up obtaining a sense probe; on the other hand, though, if we used the T7 polimerase, since our gene would "seem" to be turned around (in the opposite direction) that would already be our non-coding strand and the polymerase would do just a negative copy of it (aka:antisense probe).
Is that correct? Any better and simpler explanation? Anything would be helpful!!
Sorry it is a little bit confusing but I couldn't find a better way to describe it.
Thank u.
Yes, that's the same reasoning I do when I sequence my probes. What you're saying should be correct, and you should obtain an antisense probe.
Good luck with the ISHes!
Your reasoning is correct. In the case of (a) you would use T7 to generate your in situ probe. In the case of (b) you would use SP6.
I agree, your reasoning for using T7 in Example A and SP6 in Example B is correct.
I am not professional in that, but I was live in the same dilemma, but you should know that, the RNA probe will be form from the G+1 after the 5` TATA 3` of the promoters
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