I have a plasmid that is designed for lentiviral packaging but the size of the plasmid is ~12 kb and facing some problems in the packaging step. I've been planning the cut the part that includes the gene of interest, the GFP tag, and the antibiotic resistance gene (including the promoter and polyA signal sequence). Does linearization could be an option and provide stable integration as suggested?
Also, how does sticky or blunt end creates a difference in linearized plasmid integration?
Although its not necessary, linearization improves your chances of getting proper integrations with big constructs. With the whole, intact plasmid there are chances for partial integration of your GOI with the resistance cassette intact. This is because you can never know where the break point will be in the circular plasmid. These cells will show up as false positives. If you cut out the nonessentials sequences in your plasmid and retain only the regions that you want to integrate, you can improve the chances of desirable integrations.
Additioanlly, in case the ends are sticky, you can dephosphorylate the linearized sequence before transfection. This prevents multiple copy numbers of the sequence getting integrated. If possible, try to go for blunt end digestion but I know some labs that do sticky ends without dephosphorylation and have no problems getting the desired integration.
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