RF-Cloning.org Forum

Hi Steve,

Thank you so much. I use XL1 competent cells made in our lab for the transformation. We use them for all our cloning and a positive control transformation usually gives us a lawn growth. For example, the parent plasmid was cloned using golden gate assemble with 100 ng of plasmid and I had several (~50) colonies even with just 10% of the transformation plated. I could borrow comp cells from another lab to try though.
The PCR conditions I am using are exactly as described in the project link-
121 ng Parent plasmid, 150 ng insert
4 uL phusion buffer, 0.4 10 mM dNTPs and 0.2 uL phusion pol made to a total of 20 uL
2-step : 98 30s, 98 8s, 72 3:38 min (15 cycles) and 72 5 min final extension.
3 step : additional annealing at 60 for 20s