4 replies to this topic

[Deba_Bio]

Hi,

 

I wanted to transfect linearized plasmid DNA. I am planning to digest around 200ug of DNA in a volume of around 200-300ul. The DNA is highly AT rich so I do not want to expose it to UV. Can I directly proceed to the phenol chloroform isoamyl alcohol step, without gel eluting the DNA?

[bob1]

What you could do is set up a control experiment - digest the same amount of DNA under the same conditions in a different tube, run on gel and see if it is all linearized. If it is then you can proceed with the purification. On a side note, it is often easier to digest in multiple smaller volumes (this also means you can use one smaller volume for the control), for some reason these reactions often don't scale up as you might expect.

[Deba_Bio]

ok thanks. I would set up around 50ug digestions then.

Would column purification give any special purity over phenol chloroform and ethanol ppt?

[bob1]

As long as the PC/ethanol is done correctly, then either should work well. However, check the column limits as to how much DNA you can purify at one time, many are limited to 10 ug/extraction. Incidentally, 200 ug is a massive amount, and keeping linearized DNA can sometimes result in spontaneous re-circularization. I have always found it best to use freshly linearized DNA (within a week or so) for transfections.

[Deba_Bio]

Thanks a lot! I will transfect the plasmids within a day from linearization, so I hope that doesnt give them enough time to recircularize.