Will mycoplasma contamination lower transfection efficiency? - (Jul/14/2010 )
Curtis on Jul 21 2010, 11:28 PM said:
thanks guys,
my viral gene has a BH3 domain that is similar to Bax pro-apoptotic proteins and I have a lot of dead cells 16hrs post infection. my boss also says that this could be the reason i'm not seeing much efficiency. because the moment the gene is expressed it'll trigger apoptosis.
endotoxin could be another problem to consider. but i usually incubate 16-18hrs and also prepared fresh phenol/chloroform! so why is this happening?
i'm running another IP now,hope it works this time
my viral gene has a BH3 domain that is similar to Bax pro-apoptotic proteins and I have a lot of dead cells 16hrs post infection. my boss also says that this could be the reason i'm not seeing much efficiency. because the moment the gene is expressed it'll trigger apoptosis.
endotoxin could be another problem to consider. but i usually incubate 16-18hrs and also prepared fresh phenol/chloroform! so why is this happening?
i'm running another IP now,hope it works this time
Well..a pro-apoptotic protein.... is expression of this protein under an inducible promoter which isn't leaky?
The freshness of the phenol/chloroform does not influence the purity of the DNA sample it produces. Purity is influenced by the number of rounds of phenol/chloroform extractions. However oxidized phenol will damage DNA. If anti oxidizing agent 8-hydroxyquinoline is used, the phenol/chloroform solution will last a lot longer.
16-18hr incubation....is this the amount of time it takes before the cells start dying?
-perneseblue-