Plasmid Cleanup - Advice on how to further clean up plasmid created by alkaline lysis an (Aug/22/2007 )
Hello!
The search didn't turn up anything - so if this has been disussed pls point me in the right direction.
I have loads of a plasmid I prepared by alkaline lysis (no kits involved). The qualitiv was fine for sequencing/PCR etc.
Now I wanna use it for transfection? Any ideas for further cleanup? I was thinking about using a Qiagen-Tip, but all the protocols involve bacterial lysis - I already have the plasmid!
Any suggestions?
The search didn't turn up anything - so if this has been disussed pls point me in the right direction.
I have loads of a plasmid I prepared by alkaline lysis (no kits involved). The qualitiv was fine for sequencing/PCR etc.
Now I wanna use it for transfection? Any ideas for further cleanup? I was thinking about using a Qiagen-Tip, but all the protocols involve bacterial lysis - I already have the plasmid!
Any suggestions?
what is you plasmid DNA A 260/280 ratio?? if the ratio is between 1.8 to 2.2 then you have pure DNA and you can use it for transfection
The search didn't turn up anything - so if this has been disussed pls point me in the right direction.
I have loads of a plasmid I prepared by alkaline lysis (no kits involved). The qualitiv was fine for sequencing/PCR etc.
Now I wanna use it for transfection? Any ideas for further cleanup? I was thinking about using a Qiagen-Tip, but all the protocols involve bacterial lysis - I already have the plasmid!
Any suggestions?
As to my experience, sequecing needs higher purity that transfection. since your plasmid worked well for sequencing, it should work well too for transfection.
also as answered by last post, if 1.8-2, then OK.
by the way, I used QIA quick gel extraction kit to purified my DNA. but the final concentration is lower than what I need. so I use the same volume of buffer to elute all columns. the purity obtained is good for transfection too.
there are endotoxin free kits that will bind any endotoxins in your plasmid prep. Passing your plasmid DNA through such a column, will give you very clean DNA.
However lacking that, for Zebrafish micro injection, my lab has gotten away with just running the the DNA prep through a Qiagen gel extraction column. The resulting plasmid DNA (with a significating lost of DNA concentration) is clean enough for micro injection.
For mammalian cell tranfection, in the context of gene targeting, cleaning the DNA prep with phenol/chloroform and PEG percipitation, give good enough.
you don't need endotoxin free kits (more expensive). regular kits are good to.
What i did is a classical column for plasmid purification (qiagen, nucleospin) or matrix (QiaEX II).
I loose DNA, but it's cleaner.