Hi Everyone,
I'm trying to tranduce MEL cells with lentivirus to knock out some genes with CRISPR Cas9 and select with puromycine. This is my first time working with this type of cells and lentivirus transduction. I found that people are using Spinfection with polybrene or Mixing techniques, but I don't know which way is better. Also, how can we calculate the titer and multiplicity of Infection ? I think it is more difficult to transduce MEL cells than 293FT cells. Could you share a working protocol for MEL cells transduction? Thank you for your helps
1. You need to measure your viral titer, in this case you need to you real-time PCR method. Check taq-man probes for lentivirus LTR, or you can design primers for your construct. Use different dilution of your viral vector to make a standard curve. Run RT-PCR with your serial diluted viral sample. Based on standard curve you can calculate the titer.
2. you can use GFP-virus to test which MOI is the best for infection of MEL cells. For GFP, it is much easier to measure titer.
3. Based on expt 1 and 2 you can find the best MOI for your experiment.
4. For lentivirus, polybrene is needed (8-10ug/ml). Spininfection 2000rpm 45 minutes also help.
Good luck!
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