1 reply to this topic

[SamR]

Hi!

I am starting a new project, which involves doing a lot of in situ hybridisation on whole mount embryos, to identify genes responding to specific chemicals. I am completely new to ISH and am somewhat confused about how to design a probe. I thought to amplify a part of the gene and clone it with TA-cloning into a plasmid between SP6 and T7 sites.

My first question is, which part of the genes should I choose? Is only UTR OK? For small genes, could it be the whole gene, including both UTRs?

Secondly, am I correct by assuming that the reading frame does not matter? The START and STOP codons do not matter, do they?

Finally, what about controls. I plan to start by making around 40 probes. Should all of them have a sense strand control or would just one suffice? Any better ways to verify probe specificity?

Thanks in advance!

Sam

[Lusen]

SamR, on Feb 19 2009, 12:39 PM, said:

t to amplify a part of the gene and clone it with TA-cloning into a plasmid between SP6 and T7 sites.

My first question is, which part of the genes should I choose? Is only UTR OK? For small genes, could it be the whole gene, including both UTRs?

Secondly, am I correct by assuming that the reading frame does not matter? The START and STOP codons do not matter, do they?

Finally, what about controls. I plan to start by making around 40 probes. Should all of them have a sense strand control or would just one suffice? Any better ways to verify probe specificity?

Thanks in advance!

Sam

Just a few comment. You dont need to clone your gene, just add your SP6 or T7 with the primers and run a PCR on some cDNA

Reading frame doesnt matter and yes you should design an sense for every probe