I'm trying to create a deletion mutant in a phage with a recombineering strategy. I'm having trouble visualizing the design of the ssDNA construct/substrate to be electroporated into the cell. Would the construct consist of two "arms" that demonstrate homology to the gene to be deleted? What would be in the "middle" of the construct? Are there any user-friendly bioinformatics tools that I could use to design the construct? And lastly, is this oligo routinely ordered through a scientific vendor (eg., IDT?)
*Please also note that I can't insert any gene (ie., antibiotic resistance cassette), as phage genomes aren't large enough to support this.
Thanks in advance!!
The simplest way to think about it is to design the oligo to have the final sequence you want. So the two arms that join up in the oligo normally won't have anything in the center. The problem with not having an antibiotic selection is how will you find the mutant? Do you have an easy screen or selection to find the mutant you want? Otherwise it might be hard to identify.
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