Hi everyone, I want to order this plasmid (https://www.addgene.org/78311/) to Addgene, but when I review it, I can not find the promoter region of ampicillin resistance, so I deduce that the plasmid does not has (I already did alignments and I already reviewed the sequence and nothing).
My question is, can I subclone said plasmid in competent dh5 alpha bacteria using ampicillin as the selection antibiotic?
Thank you very much in advance! :D
The website that you gave the link for states that this plasmid can be grown in DH5alpha cells with ampicillin.
Yes, I've already read it, but when I realized that the promoter for ampicillin is not there, it causes me a lot of doubts.
Yes, it should work, it has the ampicillin resistance gene (ampR) in the construct map.
Bprom (http://www.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb) predicts a promoter ~100ish base pairs upstream of the amp gene:
- -10 box at pos. 118 ttttagtat Score 55
- -35 box at pos. 101 atgaca Score 36
Addgene has to either subculture or transform any plasmid they get and I doubt they would use puromycin instead of ampicillin given the chance. The cassette is a little unusual and poorly annotated though, so I can see why you'd be hesitant. You can always email Addgene and ask them directly though to make sure, I'm guessing the strain history is written down somewhere.
Erwin, when I read the map of a plasmid in Addgene or in any other plasmid Database, they always put the promoter and terminator for your genes of interest, but they never write at the plasmid map, those for bacteria resistance, as AmpR. So, I suppose the all Amp gen secuence include the promoter too, or if not, the promoter secuence is the ~200-500 bp before the gene
Yeah, definitely. The plasmid can be subcloned & maintained in presence of ampicillin as the vector map. Its true that the regulatory sequences for the cassettes other than the gene of interest to be cloned are not mentioned in many vectors. Go ahead.
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