Direction of Amp cassette - (Oct/17/2005 )
Hi I wish to replace a region of a large gene with an ampicillin cassette. Does the direction of the cassette matter?
of course if it's a transcribed region...
Need to transcribe the correct strand. MOst mpas show the direction of the promoter, use this to figure out what strand you need to have your gene.
If I PCR in the promoter of the Amp gene does the direction still matter? If it does I guess I should orientate it in the direction that the Kan and Gent cassettes are in?
Thank you for your help!
If you clone it with the promoter, it can go either either direction.
agree with TAP14.
If it is truly a cassette, and carries with it all required upstream regulatory sequences, then orientation doesn't matter -- it'll achieve the mutation by virtue of insertional disruption of the gene, and it'll be transcribed and translated on its own to provide the selectable phenotype...