4 replies to this topic

[verves11]

I would like to really know wheather is necessary to filter plasmid DNA before transfection. I would like to perform some transfection assays. I plan to use commercially available kits for palsmid isolation. Just going through the manual, there is no remark about filtration of palsmid DNA before performing transfection? So is this step necessary or not? I would normally diltute my isolated palsmid for recommedded concentration and finally filter trough 0,22 filter? Could this step somehow destroy the DNA?
How to exacly prepare the palsmid DNA for transfection?

Edited by verves11, 19 May 2009 - 11:03 PM.

[bob1]

I don't filter my DNA, and I have only had one infection of bacteria from transfection.  If the DNA has been prepared in a suitably clean environment, it should be sterile.

[verves11]

bob1, on May 21 2009, 03:19 AM, said:

I don't filter my DNA, and I have only had one infection of bacteria from transfection.  If the DNA has been prepared in a suitably clean environment, it should be sterile.

I am quite limited with the lab space so I have to purify my DNA in the same lab, where all the manipulation with bacteria are done. So I would be little bit worry not to furtehr contaminate my cells.

[little mouse]

I never filtered my plasmids. it was most transient transfections, but for at least 3 weeks I never had a contamination. I use medium with penicillin and streptomycine.
If you filter your DNA, I think you will lose some DNA sticked on the filter and you will not get the whole volume  of your sample back. So you might loose a lot of DNA. Think about it.

[madrius1]

Same here.

Never filtered my DNA and never got any contamination. You should be fine, specially if these are transient transfection!