I have to isolate a large plasmid from yeast and transform it in E. coli. After transformation, I often get no colonies. One reason for that is the yeast mini prep hasn't worked or the DNA concentration is too low. I tried two different isolation protocols:
From the doc: "based on the old E. coli alkaline lysis method with our Zymolyase added in the first solution."
used by Dan Gibson et. al] for their huge constructs (>100kb)
They use an expensive Qiagen large-construct column at the end, in order to purify and visualize the yeast extract on a gel, but I skip that step because the Qiagen column cost $40 per yeast clone which doesn't scale if we do many of these.
How can I troubleshoot and optimize the above-mentioned protocols? Are there any other protocols that might be more suitable for a large plasmid and don't use too expensive materials?