Hi
I am trying to electroporate a phosphorilate PCR product in my cell line. As the amount is very low, I was wondering if I can skip a purification step after phosphorilation. Does the T4 DNA ligase buffer interfire with trasfection buffer?
Thanks a lot
Hi there,
just like any buffer, it contains ions which are not recommended for electroporation. In fact you may use the ligation mix directly for electroporation if the volume is low compared to the volume of cells. I never use more than 2µL of ligation mix to transform 50µL of cell suspension. And it works fine. I've already tried more and it failed with a nice electric arc
I agree with Dominique. I used 1 uL of non-purified ligation products to transform 50 uL of cell suspension. It worked well for me. You can test your transformation efficiency using 1 uL, 1.5 uL and 2 uL of ligation mix per 50 uL of electrocompetent cell suspension. I hope it works for you.
Good Luck!
- Badges
- Science method