1 reply to this topic

[Pacupe]

Hi everybody!

   I´m cloning into a low copy plasmid (pBT). I'm just using that plasmid as a vector for further manipulations in a DNA fragment because it is the only one that I found that doesn´t have the restrictions sites I'm interested in.

  The only problem is that pBT is a low copy plasmid and I want to get enough plasmid for restriction, cloning and restriction again.

  I have a protocol to improve the yield of alkaline lisis but is very time-demanding.

  I'll really thank if somebody is able to share a proctocol to do that with no wasting time.

[perneseblue]

Use a larger volume of medium to grow more cells (~500ml, depending on how much plasmid you need. ). And use rich media such as SOC. You could also use a bavelled flask and increase the shaking speed of the incubator (300rpm) to increase oxygenation and thus increase cell mass yields