I am trying to isolate BAC plasmid of size 36kb. I tried with Qiagen Miniprep kit and Zymo BAC purification kit and ran on 0.5% gel. The bands can be seen on the gel but no bands were after I did restriction digestion with 4-cutter enzyme. I also tried ethanol precipitation but there is thick band of 250kb size of I don't know what. What might be going wrong?
if a 4 cutter enzyme cut at 1/4x4x4x4 positions then theaverage size will be about 1 cut every 240 bases so there may be many bands and all so weakly fluorescing that they may be invisibles, Try cutting with a 6 cutter and do not run the gel very far so that you can see any bands that are produced and also include an uncut sample as a control
First well is uncut plasmid and second well is RE digested sample. The sencond well does not show any bands, except for the thick band at the bottom.
Your BAC plasmid DNA seems to be contaminated with RNA (band, smear at 250kb), consider an RNase treatment step. For the restriction digest, you should use enzymes, which cut out your insert (usually the ones, which were used for cloning the insert) but don't cut in the vector, this way you should obtain two clear bands vector and insert.
Before choosing digestion enzymes, it is better to first check the Nebcutter site and check the digestion sites of the enzymes. If you are sure of choosing the right enzymes, check the efficiency of the enzymes and their buffers on a different plasmid (if you are sure of the correctness of the extracted plasmid, their shearing or buffering enzymes will not work)