Hi all,
This will be a stupid question but forgive me as I haven’t got much cloning experience. I want to clone a gene of interest into the pET28a expression systems but I’m confused. On the plasmid map I have for pET28a the T7 expression region is reversed. I have read this is due to the pBR322 convention but I am unsure how this affects cloning. Is the region actually reversed in terms of sequence and if so do I have to clone the reverse compliment of the gene into the vector or do I clone it in the normal orientation i.e. ATG – TAA on the sense (top) strand of the plasmid?
Thanks in advance
Ed
Hi Ed,
don't be confused. Your 5'end and future N-terminus has to be in the proximity of the T7-promoter (ligated e.g. into the NheI site). Your 3'end will be close to the NotI site.
To make it easier for you, I recommend to use a free Software (e.g. Serial cloner) and generate in silico the reverse complement of the sequence. You will see the same plasmid but it is easier to work with. I also recommend to do your cloning in silico first.
Best,
Uwe
Hi, you can have a look at the map here:
https://www.snapgene.com/resources/plasmid-files/?set=pet_and_duet_vectors_(novagen)&plasmid=pET-28a(%2B)
I recommend you to use benchling (free for academics- https://benchling.com) for doing in silico cloning and virtual translation to confirm that the starting codon and tags are properly located.
You just need to locate the promoter, terminator and MCS regions in the vector map. Choose a restriction enz close to promoter for your fwd primer and a site near the terminator for your reverse primer in the 5' to 3' direction.
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