I need to clone a gene (2KB sequence) from one plasmid to some other plasmid which needs to have a fluorescence tag. After proper sequencing, I could find there is presence of the stop sequence of that gene, which needs to be removed, so as to express the tag. Is it possible to design a primer for cloning it, which will not have that TAA stop sequence in it but can clone the entire gene ?
Yes, you should be able to do this (keep in mind that all cloning strategies work in the planning stage, but sometimes there are unexpected complications).
When you create your reverse primer, just make sure the 5' end of the primer excludes the stop codon sequence of the gene. You will still likely need to add a restriction site (based on your new vector you are adding the gene to). Be careful when you select your restriction site and design your primer. Make sure you are keeping the gene and tag in the same reading frame and don't accidentally add a stop codon (I know someone who did that once).
Good luck!
Hi, it should not be a problem if you do not know the 5´and 3´UTR. Exclude the TAA and make sure to provide the Kozak sequence "AccAUG". Design your 5´forward primer with the appropriate nucleotides. Simplest way if possible add an KpnI site GGTACC immediately infront of the Start codon. Cheers
Amy Klocko has given a good summery of what you need to do. You can message me if you need any further guidance.
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