How to mix your plasmid? - (Jul/12/2007 )

hello everyone

i have a basic question i am sorry ,
when you are making a working dilution of any stock plasmid DNA you have how do you mix it?
i dilute with H2O and i mix by pipetting and vortex but i kept having a strange results in my transfection than others , someone here in the lab said that its wrong to mix DNA like this you have to mix by inverting the tube and centrifuge....is he right?
sorry for the simple Q but first time to work in molecular biology.
thankx in advance

---

I dilute my maxi DNA in TE and then to mix them, brief vortex and centrifuge it.

For some special large plasmids, I tap the tube well which mixes, so that the plasmid is not damaged.

Pipetting up and down gently should also be fine. Some plasmids dont like too much shearing (but only some sensitive plasmids and not all plasmids), so it might be better to invert tubes for them.

---

THANKX scolix for ur fast reply
ok so obviously my way was wrong, my collegue mentiones something about DNA breaking because of vortexing , i will try new working solution with this new instructions and hope i got my resultd.

---

hallo spanishflower,

You can dilute DNA in TE buffer or Water.
But when you want to use DNA for electroporation you have to consider that DNA has to have a very low ionic strengh. In that case TE is not recommended.

---

i dilute my DNA sample with water or TE (according to my purpose) and after adding DNA to water or TE i close the tube lid and through a gentle strike by finger in the wall of tube and then shortly centrifuge it using microfuge

---

I alwas vortex my standar plasmid, but never larg plasmid or genomic DNA.
I've never had problems.

---

thank you all

---

well i tend to avoid pipetting up and down for dilutions, as i'm concerned about large plasmids and the possibility of breaking them.
i flick the tube or vortex 30'' gently, then short spin.

---