I have cloned a transgene and promoter in plant expression vector. Cloning of transgene was confirmed by PCR and restriction digestion and its orientation has also been confirmed. Promoter was cloned downstream to transgene with the same restriction site on both sides, which is Kpn1. Integration of promoter is also confirmed by PCR. Now the issue I am facing is that I am unable to find orientation of the promoter. I have run PCR and the desired product was observed on the gel by using full length promoter primers but I am unable to confirm if my promoter is integrated in right orientation. How can I confirm this?
I already responded to this thread but something happened on RG that made my response and the response of other researchers disappear. So, this is to comment on Domenico suggestion. I agree that subcloning lowers the probability of getting mutations but it is not completely safe.
So, it depends on how stringent are the standards in the lab. In some labs where I have been you will still have to sequence your construct. I will follow these standards. I have even received plasmids from some labs or worked with other plasmids that were left by other projects and after doing the whole job you find that they weren’t correct, etc….It is relatively cheap to sequence….
They were two economists who set up a formula that made them earn a novel prize and it also made them very rich in the nineties. They had a probability of 10 exp -24 hedge against any lose. Something happened and they went bankrupt.
Dear Abdelhalim
No, actually I made same question into another section and you replied there earlier =). Could you please check that,
https://www.researchgate.net/post/Finding_orientation_of_integrated_promoter1?ch=reg&cp=re72_x_p2&pli=1&login=zammy.butt@gmail.com#view=50c080c4e24a460e0c000045
Hi Azeem,
I think there is something wrong with link. I get : Post inaccessible. Could you please check too?
I can’t access it. This is not the first time it happens to me. Don't worry Azeem!
Hi, Azeem.
The first thing I can think about is to do the sequencing (eg the simple Sanger-sequencing) using one of the primers you have used for confirmation that the promoter was integrated.
The other possibility is to pick a primer specific to the sequence of your inserted promoter and to do two PCRs: one reaction with this primer and your vector-specific forward primer and the other reaction with this promoter-specific primer and the vector-specific reverse primer (of the primer pair used for the confirmation of the insertion). According to in what reaction you obtain bands (and whether their length will be as expected) you can find out the orientation of your insert.
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