plasmid prcipitation
Moderators: honeev, Leonid, amiradm, BioTeam
plasmid prcipitation
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...
-
- Death Adder
- Posts: 65
- Joined: Thu Nov 16, 2006 3:54 pm
- Location: Portugal
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
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
---------------------------------------
You know dog spelled backwards is god.
Coincidence? I think not. . .
You know dog spelled backwards is god.
Coincidence? I think not. . .
javaid wrote:LilKim wrote: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
- Zymo Research
- Garter
- Posts: 6
- Joined: Fri Mar 09, 2007 8:29 pm
- Location: Orang, CA
You might want to try going to Zymo Research's website or calling 1-888-882-9682 x221. For a limited time we are offering free 50 prep. Zyppy™ Plasmid Miniprep DNA Purification kits.
Plus our protocols are the easiest around
[/Shameless plug]
- The kit is Pellet-Free™ (add lysis buffer directly to bacterial culture) which should save you a lot of time
Colored buffers mean error-free visual identification of complete bacterial cell lysis and neutralization
Fast-Spin columns ensure low elution volumes and zero residual wash buffer contamination
Plus our protocols are the easiest around
[/Shameless plug]
Who is online
Users browsing this forum: No registered users and 6 guests