Large scale PCR to get milligram of DNA - (Mar/02/2005 )

Hello,
my problem concern the ability to obtain large quantity (about 1 mg) of a 3,8 kb plasmid insert alone (without plasmid) for in vitro assay. I think to use PCR, but How many amplification i'm going to do? are there methods or custom service to overcome this problem?

Thanks

Why not just do a maxiprep from a large bacterial culture and then use restriction enzymes to cut your insert out?

I used to do PCR to produce an IVT template of 266 bp (much shorter than yours). My PCR protocol used 40 cycles and would produce about 10 µg of product per 50 µl reaction. Your product is about 15 times longer than mine and so if the PCR were equally efficient (how likely?) then you would get about 150 µg per reaction. This is going on molar content only and doesn't take into account the difficulties of PCR for longer fragments and any inhibitory effects of having large masses of DNA in your reactions as things proceed.

All this being said, say 10x 50 µl reactions should do it. Just PCR, purification column and A260.

I have no idea why you wouldn't do a maxi prep/RE digest for this type of thing.

-Matt

There are, in fact, several applications in which it is better to use PCR over cloning and bacterial prep. This being said, they tend to involve much shorter fragment of DNA, normally no longer than 200-300 bp.

Those techniques that are used to investigate the sequence specificity of drug-DNA interactions are such examples. Having a flanking restriction enzyme site/sticky end can alter the binding properties and preferences of a molecule binding close by. Much of the time it is better to simply leave these off altogether.