Hi everyone - we’ve been using the NEB Q5 mutagenesis kit but would like to make our own KDL (kinase DpnI ligase) mix and buffer. Does anyone have a proven home-brew recipe for these?
This protocol always works in my hands, though it omits the DpnI digestion step.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC328208/pdf/nar00090-0243.pdf
Hi Abby,
Our DIY mix works fine - pooled enzymes to 4u/ul DpnI, 4u/ul PNK and 16u/ul T4 ligase (NEB).
I use 0.5ul of this on 0.5ul PCR product (26 cycles, Phusion) in 1x ligase buffer. I’ve not fiddled around to find the limits (this is probably slightly lavish on enzymes).
The only issue I’ve noticed is a lowering of competent cell viability, due (I suspect) to detergent carry over from the PCR buffer.
NEB makes a detergent-free Phusion buffer, but I’m ok with plating half of a 500ul SOC recovery for low hundreds of colonies.