Artur is right. Your uncut plasmid is supercoiled, which affects is passage through the agarose. In fact, you probably have multiple bands in the uncut plasmid lane, representing various supercoiled states. The "sizes" on the ladder that those bands line up with have to do with similarities in mobility, rather than length. When you linearize the plasmid by cutting it with your restriction enzymes you are able to see its actual linear size. Does your ladder really have a 12 kb mark? Or are you estimating because its larger than the highest band on a 1 kb ladder (with sizes ranging from 200-10 kb)? Keep in mind that the relationship between size and position is non-linear when you get above about 3 kb, so if its running above the last band on the ladder you won't be able to estimate its size accurately. How do you know the size of the plasmid in the first place? Are you positive that the map is correct? It's always best to double check new reagents by performing diagnostic digests with multiple different restriction enzymes to make sure they produce the expected fragment sizes. Also, when performing a double digest, perform single digests with each enzyme along side to verify that they are both cutting the plasmid individually. With a 100 bp insert, it's unlikely you'll be able to tell the difference between singly cut and doubly cut plasmid.

Artur is right. Your uncut plasmid is supercoiled, which affects is passage through the agarose. In fact, you probably have multiple bands in the uncut plasmid lane, representing various supercoiled states. The "sizes" on the ladder that those bands line up with have to do with similarities in mobility, rather than length. When you linearize the plasmid by cutting it with your restriction enzymes you are able to see its actual linear size. Does your ladder really have a 12 kb mark? Or are you estimating because its larger than the highest band on a 1 kb ladder (with sizes ranging from 200-10 kb)? Keep in mind that the relationship between size and position is non-linear when you get above about 3 kb, so if its running above the last band on the ladder you won't be able to estimate its size accurately. How do you know the size of the plasmid in the first place? Are you positive that the map is correct? It's always best to double check new reagents by performing diagnostic digests with multiple different restriction enzymes to make sure they produce the expected fragment sizes. Also, when performing a double digest, perform single digests with each enzyme along side to verify that they are both cutting the plasmid individually. With a 100 bp insert, it's unlikely you'll be able to tell the difference between singly cut and doubly cut plasmid.

To clarify, the Uncut plasmid is running normally. I have been working with it for some time now and have verified its size. It ran at 6kb, as expected, with some blurring from supercoiled states. I did previously run diagnostic restriction digests, with these and other enzymes and I have selected.

The digested, linearized (based on the shape of the band) plasmid is the one that ran far too big. 12 kb is basically an estimation, it is just slightly above the 10 kb mark in a spot which the accompanying guidelines for the ladder suggest represents 12 kb. But even if this is not to be considered an accurate measure of size, the size is still way off what I would expect.

@Stephan: If it's worked in the past, I'd suggest 1) repeating the digest with single enzymes to make sure they're still alive and 2) using less plasmid in the digest. Major reasons I've had unexpected sizes from plasmid digests in the past have to do with incomplete digest due to enzymes that are losing their efficiency, supercoiling due to too much uncut DNA left in the digest, and contamination in the plasmid prep with ligase that hasn't been properly inactivated (which may be resulting in strange ligation patterns or at least protein binding to your DNA, which will alter its size). Re-purifying the plasmid using a column to remove any contaminating proteins would be a good first step. Check it's purity by spec (nanodrop), focusing on 260/280 and 260/230 ratios (http://www.bio.davidson.edu/projects/gcat/protocols/NanoDrop_tip.pdf). Then perform side by side single digests with Not1, Sal1 and the double digest with Not1 and Sal1. Leave the reaction for longer than an hour (even overnight at RT will be fine with these enzymes).

The other possibility is that your plasmid has undergone some kind of rearrangement event during passage through cells. Check the map for repeated stretches of sequences or highly repetitive sequences. In rare cases, these can create plasmid instability, which can wreak havoc on restriction analyses.

Worst case scenario, I'd send your plasmid for sequencing to make sure it's still the same plasmid that you started with.

Good luck!

@Artur, no offence was intended. Some of my initial terminology and phrasing was surely unclear.

@Elizabeth, thank you I will try some of your suggestions.

You need a positive and a negative control to fig out what happened in your product