I have a 4.1 kB plasmid with insert I want to linearise and use as a PCR standard subsequently. I've got minipreps around 170 ng/ul and preparing midiprep.
I found a single cutting enzyme we have, BglI. Problem may be that the batch we have was assayed in 2001, so it's a bit old.
I need to get ~100% pure linear plasmid. It shouldn't be shredded or degraded.
So first I try if the enzyme works at all on a small amount, like 2 ul of plasmid.
But for the final product I need to cut a large amount of plasmid, say 20 ug in total.
Is there a way to calculate how much enzyme I need for 20 ug of plasmid (let's say 2x10 ug in a 100ul reaction), when the NEB website says:
It also says the enzyme is suitable for extended digestion. So it's better to use more enzyme (if we have enough) for 1 or 4 hours, or digest overnight to get a completely cut sample? Will it increase a degradation or nuclease activity if I digest it overnight?
Next thing is how can I be sure it's all cut? If it is, I can purify the product using phenol-chloroform extraction rather than purification Qiagen kit (they only define they're kit for 4kb long products, which is a borderline). But if it's not I would need to somehow dilute it (not to overload the gel) and gel purify from several lanes with the Q kit.
I heard something about good and bad cutters, is BglI a good cutter or not? I have several other options (XmnI, NcoI or even BamHI, but I not prefer that one, since it's quite close to the insertion site).
Do you have any comments to this? Anything will help. Thanks.