plasmid prcipitation

[javaid]

i have isolated plasmid and is finally suspended in TE but i need it be dissolved in PBS coz i need it for transfection, so may i know how to precipitate the plasmid. do i need to add salt...

[LilKim]

1. precitate in 100% ethanol fow at least 1 hour (or overnight) at -20.

2. spin the sample (pellet the DNA)

3. decant supernatant.

4. wash with 70% etoh.... {spin the sample (pellet the DNA)}

5. decant/remove supernatant

6. allow DNA pellet to air-dry

7. resuspend in PBS

[javaid]

[quote="LilKim"]
but i think adding salt will enhance the precipitation.. will it ppt without salt.. n if not then which salt to use n conc. thanx

[SororSaudade]

You should also add sodium acetate (1/10 of the final volume). I think this is just to stabilize the plasmid and not to improve percipitation, though I'm not quite sure.

I generally use for transfection plasmids dissolved in TE... is there any advantage in using PBS?

[Vagabond]

Actually with the above method your are ppt the plasmid with the EthOH not salt.

If you are referring to the use of PBS at the end that is just your physiological buffer.

I usually resuspend my plasmids in ddH2O pH'ed to around 7. But that is me. I see no reason that you can not resuspend it in TE for transfections (depending on your transfection method). Lipofectimine should be fine (you will dilute out most of the TE) now if you are using electroporation well then yes even PBS can be an issue, arching. More info is required, cell type, culture conditions, transfection method, prep protocol, concentration, amount to be transfected, etc.

GL

[LilKim]

but i think adding salt will enhance the precipitation.. will it ppt without salt.. n if not then which salt to use n conc. thanx

yes.. you're correct... use 10% sodium acetate (of your pDNA+100%EtoH solution) and precipitate the entire solution at -20.



sorry, about the confusion

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