plasmid prcipitation

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    • #6998
      javaid
      Participant

      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…

    • #69287
      LilKim
      Participant

      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

    • #69291
      javaid
      Participant

      [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

    • #69292
      SororSaudade
      Participant

      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?

    • #69303
      Vagabond
      Participant

      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

    • #69335
      LilKim
      Participant
      quote 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

      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

    • #69868
      Zymo Research
      Participant

      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.

      • 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]

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