We performed a transformation-associated recombination protocol in yeast of lot of DNA fragments and the vector to get a YAC of aprox 35KB. We first checked the proper assembly of the fragments by multiplex PCR of general DNA extraction of several yeast colonies. Once we observed 100% of the tested colonies had all the correct junctions we moved forward to do maxiprep to get just our YAC. To do so we adapted a normal maxiprep for bacteria (Zymo) just preparing a typical P1 RESUSPENSION BUFFER with zymolyase to incubate yeast cells 1hr. After this step, most of the adapted protocols just follow the normal procedures of the manufacturer. Running it on a gel showed an approximate size to what we expected. Next we did a `diagnostic digestion´ with any random restriction enzyme to check our product and Surprisingly, none of 3 enzymes worked. We check RNA contamination but it was not a problem. I tried multiplex on this product and it worked perfectly. Ruling out options my last concern was genomic contamination and, when I treated with exonuclease 5, the signal almost dissappeared. This, in addition to sequencing and transformation failure of this product make me think of a huge contamination of my YAC with genomic DNA so, am I right? and more important, how can I fix it?

Sory for the long text, thanks