Need to run gel after miniprep before digest? - (Aug/04/2016 )

In what cases do you and don't you need to run a gel to purify a plasmid after a miniprep? When is it okay to go straight to the restriction digest after a miniprep, without going through electrophoresis and gel extraction?

What about all the natural, nontransgenic plasmids that get caught up in the miniprep? They aren't removed until electrophoresis, right?

Thanks

naglemi on Thu Aug 4 05:37:37 2016 said:

In what cases do you and don't you need to run a gel to purify a plasmid after a miniprep? When is it okay to go straight to the restriction digest after a miniprep, without going through electrophoresis and gel extraction?

What about all the natural, nontransgenic plasmids that get caught up in the miniprep? They aren't removed until electrophoresis, right?

Thanks

It's always ok to go straight to the digest, if you are prepping single colony minis.  I've never done otherwise.

So you transform your ligation/chewback.  Pick single colonies into minis.  Miniprep those minis.  Digest all of them individually to ensure that the insert is present (with an enzyme which preferentially does more than just pop out the insert).  And as long as the gel of those digests shows that particular minipreps are pure and insert containing, you're good to go with those minipreps.

Since the nontransgenic plasmids and the transgenic plasmids carry the same replication origin, odds are that they will not be present in the same colony - unless the insert is unstable and the plasmid is in the process of breaking down.

If you electrophorese before a miniprep you will see multiple bands, but only because the transgenic plasmid is in supercoiled, relaxed, and multimer forms, not because it's contaminated with non-transgenic plasmid.  A restriction digest (to completion) will convert all of these forms into the same form.