Although the transfection and infection were successful using another transfer plasmid, with PWZL.hygro HRAS G12V we didn't get a good infection on MCF10A cells.
We used the 3rd generation of lentivirus production, as following; 2.2µg GAG-pol as packaging plasmid, 1.2µg VSV-G as envelops plasmid, and 3.3µg of transfer plasmid on HEK293T cells.
can you help me!? any tips?
Thank you in advance
Hi Abrar,
1) Play with different ratios of 3 plasmids
2) use Positive and negative controls such GFP reporter
3) make sure you have good titre, use ELISA kit
4) try adding different concentration of viral supernantant, 10%,30%,50% and 100%. to target cells
5) make sure constructs are working and inframe.
Good luck.
Anand
Thank you Anand for suggestion , we will try to different ratios of the plasmids and see the result hopefully it will work !
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