You can use te band size to help you determine the restriction map. In your case, you may want to run 3 different RE digests.

1) Digest by XhoI and PstI
2) Digest by XhoI and BamHI
3) Digest by PstI and BamHI

Let’s say the following are the band sizes of each RE
1) 1kb, 4kb
2) 2kb, 3kb
3) 0.5kb, 4.5kb

By comparing result 1 and 2, you can basically tell that the cut site of XhoI is nearer to PstI than BamHI because the RE digest product is smaller (1kb).

By comparing result 2 and 3, you know that the cut site of BamHI is closer to PstI than XhoI because of the digest product being smaller (0.5kb).

So, the result would be XhoI, PstI and then BamHI (in this example).

Do you have a photo of a gel, or a schematic diagram, or something that shows what bands you get when the plasmid is digested with one or more enzymes? I’m guessing you do (if not, you can’t generate a map).

I’ve linked to a web site that gives an example similar to what I think you’re dealing with. Hopefully that will help get you started.

(Note that the link uses a hypothetical plasmid of only 50 bp. In reality, no plasmid is that small. Don’t be worried if the plasmid in your problem is several thousand bp – that’s normal.)

Unfortunately, the example doesn’t address the situation where “some bands can conceal multiple fragments.” That just means is that a digest might result in two (or more) DNA pieces that are the same size. If so, they will appears as a single band on the gel.

To check for that possibility, add up the sizes of all the bands shown in each lane. The total should be the same for every lane (equaling the total size of the plasmid). If one lane has a lower total, figure out how much it’s “missing”. Chances are, you’ll see that there is a band of that size in that lane. That band probably represents two different fragments that run together. In other words, if you count that band twice when you add up the sizes for that lane, you should get the correct total size. (It’s also possible to have two or more doublet bands, or triplet bands, etc., but hopefully your problem isn’t that challenging.)

Your not getting answers because this is a difficult question. I am an evolutionary biologist, but I see no functional answers to this question.

Good luck. ( BAMHI restriction site—- G’GATC_C—- Leaves a sticky end )