Hi,
I have a puromycin casette transfected into an integration defective lentivirus vector. Normal (integration competent) lentivirus titer assays expects the user to count surviving cells after 3 days.However, I am afraid the plasmid will be diluted by then and the titer will be inaccurate.
I am investigating IDLVs so using integration competent vectors is strictly forbidden!
Is there a way around this problem?
hi see this link is helpful
https://www.systembio.com/downloads/web
Hi Abel
I would recommend that you titre IDLV by qPCR. There are some good protocols here:
http://www.bushmanlab.org/tutorials/pcr
If you harvest genomic DNA from transduced cells 2-3 days after transduction, then you will get an accurate titre, using the described Late-RT qPCR assay.
Puromycin selection isn't very suitable for IDLV vectors. IDLV will integrate with 0.1-1% efficiency, so you would effectively purify integrated proviruses. From your project description, it sounds as though this would be a problem.
If you want to purify cells that have been targeted by vector, replace Puro with GFP and simply sort GFP-positive cells 2-3 days after transduction.
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