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Poll: linear vs. supercoiled, blunt vs. overhang?


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Poll: Poll: linear vs. supercoiled, blunt vs. overhang? (1 member(s) have cast votes)

Linear vs. supercoiled?

  1. Linear (1 votes [100.00%])

    Percentage of vote: 100.00%

  2. supercoiled (0 votes [0.00%])

    Percentage of vote: 0.00%

  3. WTF? (0 votes [0.00%])

    Percentage of vote: 0.00%

Blunt vs overhang?

  1. Blunt (1 votes [100.00%])

    Percentage of vote: 100.00%

  2. 3' overhang (0 votes [0.00%])

    Percentage of vote: 0.00%

  3. 5' overhang (0 votes [0.00%])

    Percentage of vote: 0.00%

  4. dosen't matter (0 votes [0.00%])

    Percentage of vote: 0.00%

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#1 jah

jah

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Posted 25 October 2009 - 07:34 PM

Going to be Electro-transfecting some primary chondrocytes shortly with the goal of stable transfection of an shRNA-expressing plasmid to my GoI under either G418 or Puromycin selection. I've optimized my electroporation technique for transient transfection with supercoiled DNA, though I only get about 30% up-take. Hence, my motive toward creating a stable line thus is to kill off the non-transformed cells that overwhelm the sensitivity my downstream assays.

Generalizing from what I've read on the topic, it would appear that linearized plasmid DNA is most efficient for stable transfection, and false-positives and off-target effects seem to be minimized. Even less clear from what I've read is whether the ends of the cut DNA make much of a difference? It seems to me that an overhang would be less efficient in integration, but also less likely to have off-target effects, whereas a blunted pDNA would suffer (benefit?) oppositely? I saw another suggestion - not sure I understand the logic - which said to add the complete digestion mix - without heat-inactivation(?) - directly into the electroporation buffer. I'm a little leary of what the RE buffer will do to my cells.

Any thoughts or experiences you'd care to share?




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