Please recommend a way to label intact plasmid DNA with something to direct detect it in cell after transfection. Better to have enough sensitivity to detect single plasmid DNA.
Single-copy detection is likely to require an amplification step. Detecting a single-copy plasmid would be alot like detecting a single-copy gene, which can be trivially done via PCR or qPCR.
There are also roundabout ways of detecting a single-gene via florescent labeling using a florescent DNA binding protein specific for sequence. I can't seem to find the reference, but it worked like this:
-The trasngene was enginered to have multiple sequence motifs (e.g. UAS) that would be recognized by a florescent protein (e.g. GAL4).
-The transfected cells would express a florescent DNA binding protein that would bind that sequence (e.g. GAL4-GFP).
-In cells where the transgene was present, it could be detected as a focus of signal.
-In cells where the transgene was absent, the GFP signal did not have a focus.
Do you want to detect the integrated plasmid in your cells? Depending on it's size you could try to do FISH, or add a fluorophore-tag to your construct and check the transfection efficiency by FACS.
Thank you, Brain and Sabine,
Brain, do you have more direct method?
Sabine, the main goal is to track and quantify plasmid DNA get into the cell. Sensitivity to single plasmid DNA is preferred, but not absolutely needed.
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