we have to transfer a kan resistance fragment to a zeo resistance fragment in a vector for cloning. This fragment can only be cut with BclI and EcoRV. Because of that, we have to make a dcm-, dam- vector.
After the transformation, we use machery-nagel kit to make plasmid DNA.
The problem we've got is that we have very less yield and when we do a digest the DNA is broken down.
We grow the vector on 37°C for the plasmid extraction in 200 ml. We use also lysozym before the lysisbuffer. After neutralisation we centrifuge 15 min by 4000rpm. Than we follow the rest of the low-copy plasmid protocol.
We get concentrations of max 250 ng/µl.
We do first the digest with BclI at 50°C just 1 hour and than 1 hour 37°C with EcoRV. After checking on gel, there's nothing but smear. Do we only put the DNA by 50°C and 37°C, the DNA stay stable.
What can we do to get a higher yield and DNA that is stable enough for digest?