Losing DNA in purification?

Question

I asked a similar question about a week ago, thanks for the answers, but what I really need to know is if I can go from digestion to dephosphorylation to ligation WITHOUT a purification step in between. I am using Promega Xba1, Roche CIP and can either use Invitrigen or NEB ligase. The vector is about 14kb long and my bugs don't seem to like it! I get after a mini, about 40ng/microlitre, so I can't afford to lose anything with purification. I can't afford to run any on a gel to see if anything has worked. I've tried gel purification with columns, cleaning through a mini prep column, even the old style 'freeze squeeze' but each time I lose too much. Is purification NECCESSARY and if not, does anyone know if the buffers will be compatible?

Answer 1

I always did the dephosphorylation simultaneously with the restriction. I used NEB restriction enzymes and shrimp alkaline phosphatase (SAP) from USB. As long as the total volume of enzymes as less than 10% of the reaction (to avoid too much glycerol in the mix) it worked fine.

You can heat inactivate XbaI. Also SAP is heat inactivated (though I haven't tried it). Check if CIP can be heat inactivated. If it can then probably you can get away with heat inactivation of the mix before using it for ligation. If however you have residual phosphatase activity then ligation will fail.

It looks like you have a medium or low copy number plasmid, but anyway since it is so big it sticks very well to the column.
The best choice would be to prepare lots of plasmid and afford a single purification step, just before doing the ligation.
Why don't you just use bigger culture volumes to get more plasmid? You could either do a midi or maxi prep, or use >1 mini-prep columns.

I've been working with very low copy number plasmids so I know the problem. If you are cloning a PCR product I'd suggest that you first clone it in a simple high copy number vector like pBluescript, sequence it and then subclone it to your desired "problematic" vector. Otherwise you might get problems with the sequencing reaction-I've been there. If you are subcloning then OK.

By the way, the transformation efficiency for very big DNA fragments increases a lot if you add gyrase in the mix. I don't quite remember the citation (could be nucleic acids research) but I bet you can find it on Pubmed.

Answer 2

So the problem I see here is that you're using enzymes from 3 different companies, so it's highly unlikely that they'll be compatible.

A couple of options that come to mind are:

Order all 3 enzymes from NEB (and use antarctic phosphatase instead of CIP). NEB's enzymes work in each others buffers typically, so you can do the xba digest, then dephosphorylate the DNA, then heat kill both enzymes, then add ATP and ligase (NEB's ligase works in the other buffers but needs added ATP to function).

You can also try combinatins of heat killing the enzymes followed by ethanol precipitation or drop dialysis on a 0.022 micron membrane in distilled water.

Anyhow, short answer is that trying to do those reactions with all those different buffers is going to cause trouble. At the very least you need to heat inactivate the enzymes.

Answer 3

I would take the approach of tackling the problem at a different stage i.e. amplifying the plasmid. This may well save a lot of buffer optimization and the expense of buying new compatible enzymes. Some possible hints are included below.

Try growing your bugs in a bigger culture and doing a bigger prep then precipitating the DNA into a smaller volume.

Alternatively use YT broth as a media. In this they will grow more slowly but you should get a better yield of plasmid for the same density of culture.

Answer 4

You can do all three so long as the buffers are compatible if you get the enzymes from the same company. NEB and Promega both do a buffer that is compatible for multiple enzymes. (The % activity in that buffer is shown on the data sheets for the enzymes). I think the name of it (from one of the companies) is the mutli-core enzyme buffer and it is usually provided with the enzyme. It's worth heat inactivating your enzymes between reactions.