Should I purify my ligation mixture before transforming the competent cells? Or would I be able to directly use the mixture, but suffer from a reduction in transformation efficiency instead?
Direct add the ligation mixture to the competent cells for transformation. Then follow the manufacture's instructions for competent cell transformation. You will be fine.
Hi there,
Purification/precipitation should be considered if direct use of ligation mix for transformation is unsuccesful. It is actually known that transformation efficiency is lower when using ligation mix directly.
Hi,
You can directly add your ligation mix to your competent cells. But be sure not to put too may of the ligation reaction because the buffer in the ligation mix might inihibit transformation efficiency. So for example if you have 10uL ligation reaction you can start using half of it for transforamtion and divide the half to 3uL and 2uL for another transformation so you can have varying concentration of the ligation mix.But as for me, purification is not really necessary.
Hi Avin,
It also depends on which kind of competent cells you are going to use: [1] chemically competent cells (heat-shock) or [2] electroporation-grade competent cells. With salt in the ligation mixture, if high volume of it is added to the electroporation-grade competent cells, the high amount of salts may cause electrical arcing (you will hear a loud 'pop' noise) during electroporation. Electrical arcing reduces transformation efficiency. So, some companies suggest using 1 ul of ligation mixture for electroporation.
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