co-transfection of plasmids - ratio - shRNA plamid with plasmid expres'ng target (Nov/12/2007 )
Hi,
I need to co-transfect a plasmid expressing a viral protein and a shRNA expressing vector in SF9 cell line to check for knock-down of viral protein expression. Its not an insect virus, so I can't infect the virus directly in teh cell line. In what ratio should I transfect the 2 plasmids; I realise that the plasmid expressing shRNA should be in excess, but it would be helpful if I could know the accepted ratio. Any other suggestions will also be appreciated greatly!!!!
Thanks in advance....
You will have to try out different ratios for the shRNA vector. You could try 1:3 - 1:10. And based on the results you have to decide which one works best for you.
Hi, scolix,
How long do you normally wait for expressed shRNA to take its action? It 48 hr long enough, or you would wait for 72 hrs post transfection? Thx.
Hi, scolix,
How long do you normally wait for expressed shRNA to take its action? It 48 hr long enough, or you would wait for 72 hrs post transfection? Thx.
If we cotransfect a protein and its corresponding shRNA , then 48 hrs should be fine. Like expressing luciferase or gfp and knocking it down, 48 hrs, you can see difference but we waited for 72 hrs for maximal effect.
But if its endogenously expressed, it depends on the half life of the protein. could be a few days before noticing any effect.
thanks!!! Could you please tell me what is the maximum amount of DNA that can be transfected in 1 well of a 24 well plate? I was planning on 0.8 - 1.6 ug. Is that ok? Or can I opt for less?
It depends on which reagent you use. 1.6 ug will be the high end. You need to decide base on teh toxicity level of transfected cells.
Try a range of different concentrations with your protocol and you could decide on a specific amount based on the transfection. But like genehunter suggested, have 1.6 as your upper limit.
may be with calcium phosphate transfection, some people might use higher amounts of DNA. I am not so sure.
Thanks again genehunter and scolix. I am using FuGENE 6 from Roche. Your suggedstions have given me an idea how to proceed. I am going to start with 0.4 - 0.8 and then work upto 1.6 ug. Wish me luck. Thanks once again!!
Good Luck !!!