I did a sequential digestion of ApaI and BamHI on a few of my plasmids, using 4ug each (plasmid size ranged from 4kb to 9kb), followed by gel extraction.

All but 1 of my plasmid ,the biggest one which had a size of 9kb, resulted in an absurdly low yield of 3ng/ul, when eluted in 20 ul. The rest of them had yield of at least 70 ng/ul in 20ul.

I think it is possible that the yield I am getting is also not the cut plasmid as well, as I am having trouble getting colonies after ligation and transformation.

I would like to know if anyone has an explanation to this. Is it because the plasmid is too big? I am using the gel extraction kit from biobasic and the binding capacity is supposed to be 10ug, so amount should not matter, though I'm not too sure about size.

I have done the following to troubleshoot it.

Lowering the amount to 1ug (still got only 2ng/ul)

Incubating at 37C - 50C before eluting (same yield still).

Redoing the digestion (same low yield)

Any help is much appreciated.