I have amplified glucose oxidase gene (1.8 kb) from A. niger and used XhoI restriction site for forward primer and XbaI restriction site for reverse primer and successfully cloned in vector PpicZalpha(3.6 kb).
But after getting clone in E. Coli (Top10 F’) colony PCR showing glucose oxidase gene amplicon (1.8 kb).
Meanwhile plasmid isolation and its digestion with XhoI and XbaI not showing release (1.8 kb gene and 3.6 kb vector) instead showing linearized gene plus vector(5.4 kb) .
Can anyone tell me what could be the possible problem in digestion or in restriction site?
The problem is likely to be that your XbaI site overlaps an E. coli Dam methylation site. As we used to say in my lab when using XbaI: "Dam those methylation sites!" Anyway, your solution is to use an E. coli host that is dam- . See https://www.neb.com/tools-and-resources/selection-charts/dam-dcm-and-cpg-methylation for details.
Dear professor,
thanks for your valuable suggestion.
Now I will use DH5alpha (dam-) to clone my gene.
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