I did whole plasmid sequencing of a plasmid I received from my colleagues when the result came out why was everything reversed in it? Meaning the format should be 5'promoter-gene-terminator- t7-3'. but instead, it came as t7-terminator-gene-promoter. I need to clone my gene between the t7 and the terminator. How should I clone it? Should I do everything reversed as I have to keep the format as 5'-gene-3'.
Please suggest.
Sequence assembly software usually assigns the + and - strands arbitrarily and sometimes this is different from how you would personally assign it, since the software doesn't really know what it's "supposed" to look like. If you take it into plasmid annotation software and flip the +/- strands does that fix the issue?
Alexandra Johnson Maybe yes. I will try it. I just thought what if there is something wrong with the plasmid?
The reason might be that you used a restriction site located towards the terminator region while designing the forward primer and a restriction site located towards the promoter region while designing the reverse primer. You should always opt for restriction sites located towards the promoter region for designing the forward primer and those located towards the terminator region for designing the reverse primer. Sometimes, the plasmid map and sequence available at corresponding websites are presented in reverse order, which can confuse researchers. It is possible that you encountered the same issue. Best wishes!
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