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Basic cloning question

Basic cloning question - Molecular Cloning Forum

Basic cloning question - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 08-11-2011, 09:57 AM
Pipette Filler
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Default Basic cloning question



I'd like to clone a gene + promoter into pBR322 and express it in a strain (not for overexpression purposes). I have cloned into plasmids before to overexpress genes and then it's obvious where to put the genes i.e. MCS. However, I'm a bit confused as to whether the gene will be expressed properly if I just whack them in the middle of the Amp or Tet gene. Do I need to encode a stop codon upstream of my promoter to prevent read-through. Does it need to be in the same orientation as the gene I'm cloning it into?

Thanks!
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Old 08-13-2011, 01:37 PM
Pipette Filler
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Default Re: Basic cloning question

dear
please if you explain your q. you are saying that you have gene n promoter in one plasmid which over expresses the gene now you want to clone these in to new plasmid (pBR322)?
if you want to control the sequence you need to add regulatory regions for the promotor
i think simply adding stop codon will not work
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