I want to get rid of the Pst1 enzyme site, which is within the promoter region of a gene (from a mamalian cells that i want to clone into a bacterial cloning vector first along with its promoter region) do you folk think that a single point mutation would be enough? would there be any star activity if i stick a single point mutation? did anyone expereience this with Pst1. since its not an ORF where you would like to introduce a silent mutation, is there any tip for which of the base would be the best target of the six bases of Pst1 recognition sequence or it should be just random. Thanks
Hello Sadeeq:
Yes, a single point mutation anywhere along the 6 nt PstI recognition sequence will render the sequence unrecognizable by PstI but question is, would this manipulation affect the promoter activity? If I were you, I would leave this recognition site alone and try to use other REs. If you explain little more, I may be able to help you with a better cloning strategy.
Dear Syed A ALi,
Thanks for your opinion. Basically Pst1 site start just one bas pair upstream to the presumable transcription start site, so i guess by mutating a single base would not be that drastic i suppose. secondly because of the cloning strategy for downstream procedures, replacement of other promoters and genes, i have already designed Pst1 as a cloning restriction site, so it will be a bit more work for me to leave it like that.
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