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bacterial transformation - (Feb/14/2007 )

Does anyone add PEG to the bacterial transformation by electroporation to increase the efficiency of the transformation?


no, however sometimes i dialyse my samples before electroporation and i do this dialysis with PEG.


I sometimes add PEG6000 to my ligation reactions to increase the chance that the vector and insert meet. I then perform electroporation but it has never worked:( you need to ppt your DNA before electroporation.


According to the New England Biolabs website:
Q1: What factors can cause incomplete ligation and/or transformation using the Quick Ligation Kit?

A1: The most likely causes are:
a. The Quick Ligation reaction was heat inactivated. Heat inactivation in the presence of PEG, which is contained in the Quick Ligation buffer, inhibits transformation.

b. The Quick Ligation mix was not purified prior to electroporation. PEG in the Quick Ligation buffer prevents electroporation. Ligation products can be purified using a commercially available spin column.

c. The Quick Ligation was incubated overnight. PEG in the Quick Ligation buffer may cause a decrease in transformation efficiency over time. There is no addition benefit of letting the Quick Ligation reaction go beyond 30 minutes.


Thank you all blush.gif