DNA is about 8kb, cells: XL1-blue, selection: ampicillin, PCR screening of clones: Encyclo Plus PCR Kit (Evrogen), DNA exstraction: Plasmid Miniprep (Evrogen).
Site-directed mutagenesis per QuikChange Site-Directed Mutagenesis protocol using Thermo Scientific Phusion Hot Start II High-Fidelity DNA Polymerase kit.
P.S. All kits work good (I've got this DNA with another mutations).
Normally, commercial kits for plasmid extraction, be it minipreps or midipreps, work fine if you follow the instructions. There may be a problem if your plasmid is low copy, if there is something wrong with the concentration or quality of the antibiotic for selection, or if there is strong counterselection against the plasmid due to the production of a toxic protein.
You could try increasing the amount of starting cells. Just don't go over the binding capacity of the column. Or as Khajezade Mohadese said, reduce the elution buffer volume. It also helps to warm up the buffer & let it sit on the columns for a minute in the last elution step.
I agree with Khajezade Mohadese , reduce elution buffer, check antibiotic is not out of date and fresh Amp plates always used for selection. Check your kit is suitable for extraction of 8 kb plasmid, some kits are better than others. How low is the concentration?
I agree with Katie A Burnette and Khajezade Mohadese, warm up and
reduce your elution buffer. Furthermore, I advice that you could
use centrifugation to remove the initial precipitation to obtain
more supernatant for subsequent experiments. After we adopt this method,
the final plasmid concentration is much better than before.
Check whether the amplicillin is still working. If the amplicillin is still good, try increasing the amplicillin concentration.
As Katie wrote: "It also helps to warm up the buffer & let it sit on the columns for a minute in the last elution step." In my case I preheat the MiliQ H20 (that could be elution buffer in your case) to 50 degrees Celsius. Then I place the column in the collection tube in to the thermoblock (50 degrees Celsius) for 1 minute. Then I spin it and pipette the liquid from the tube back in to the column. Then I place it for another 1 minute in to the thermoblock. Afterwards I spin it second time to get the plasmid solution. Sometimes I follow with 10 minute incubation at 95 degrees Celsius and final spin to precipitate and pelet the protein inpurities. Furthermore you can double the wash buffer volumes to increase the purity.
And for some plasmids you can use chloramphenicol (or spectinomycin) amplification to increase the yield.
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