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Here is the cropped image I am working with:

DNA Gel Electrophoresis

It doesn't really matter for my question, but this was performed with 0.8% agarose gel in 1xTBE buffer using ethidium bromide and a TriDye 1kb DNA ladder, run at 100V, 200 mA for 45 mins.

I am new to DNA electrophoresis so I am still learning how everything works. I understand that the DNA migrates a distance inversely proportional to the log of its molecular weight, and also based on the conformation it takes (closed circular, open circular, linear, etc.)

This run was meant to be a sort of mock-forensics experiment. There is DNA from the "crime scene", "suspect 1", and "suspect 2". There are 3 samples from each, one is untreated, one is digested with EcoRV, and one is digested with PstI. I thought for certain I knew the order of the samples (which were provided but simply labeled 1 - 9), but I am starting to have second thoughts. Based on the information I was given I am unsure how to interpret what is going on with each set of bands, especially since I am now doubting which sample is which. One of the goals besides empirically analyzing the data is to determine which suspect committed the crime, but after consideration I believe I am either missing something, or misunderstood the sample order.

Here is the information I have regarding the samples, as per the procedure I was following:

You will be given 9 samples for the separation and analyses. A specific region of DNA from each of the three samples (Crime scene, Suspect 1, and Suspect 2) were prepared by

  • Isolated and incorporated into plasmid (circular form) - untreated
  • Above mentioned plasmid was cleaved at the specific DNA sequence motifs – treated 1 & 2

Later, they are listed as:

Samples
Crime scene (Untreated and treated)
Suspect 1 (Untreated and treated)
Suspect 2 (Untreated and treated)

Given this information and lack of contradiction during the procedure, I assumed that the samples labeled 1 - 9 corresponded to

  1. Crime Scene - untreated
  2. Crime Scene - EcoRV
  3. Crime Scene - PstI
  4. Suspect 1 - untreated
  5. Suspect 1 - EcoRV
    .
    .
    .

As instructed by the procedure, I am 99% sure that sample 1 was placed in the well next to the ladder, and then 2 - 9 followed sequentially to the right. So samples 1-9 should be placed in wells 2-10. I only leave room for doubt as I am trying to figure out what is going on.

Moving on to the gel itself, the double banding patterns on wells 5, 8, 9, and 10 appear almost identical to what I have seen of a mixture of oc and ccc forms after plasmid prep. This is what I would guess the untreated samples should look like, assuming they were prepared in that way. All I know of how they were prepared is in the line "Isolated and incorporated into plasmid (circular form)" from the procedure. Even so, I have had no indication that the untreated samples would be grouped together, or that they would be samples 7, 8, and 9. I guess anything is possible at this point though.

It was my understanding that digestion with a restriction enzyme causes a break in both strands leading to exclusively linear form. Linear form should travel less distance than the ccc form, but more distance than the oc form. So even assuming I got the samples completely mixed up and 8, 9, 10 are the untreated samples, I don't see how the rest of them could be a result of being linearized.

I also have no idea how to make sense of the three bands in well 2, I haven't learned anything that would explain that given what was supposed to happen. I actually don't have any idea how to make sense of the whole first set of samples (#'s 2, 3 ,4) seeing as all of their bands are significantly lower than every other band on the gel.

Sorry for the wall of text, I am just very confused as to how to piece this all together. What could describe the patterns shown given the context I provided?

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1 Answer 1

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You would think that the plasmid of suspect 1 and 2 is about the same size (or exactly the same size), and that the plasmid found at the crime scene is identical in length and sequence to one or the other. Finally, we have to assume that the DNA of the two suspects are different from one another.

Lanes 5-10 look like undigested plasmid and carry little information.

[OP] It was my understanding that digestion with a restriction enzyme causes a break in both strands leading to exclusively linear form. Linear form should travel less distance than the cc form, but more distance than the oc form.

The restriction enzymes cut both strands. If there is one site on the plasmid, it gets linearized and you can measure the size of it. If there is more than one site, the plasmid gets fragmented. The sum of the fragment sizes should match the total size (here are some examples). There is no example of a full-size linearized plasmid on this gel, so no bands between cc and oc form of the plasmid in this example.

[OP] I also have no idea how to make sense of the three bands in well 2, I haven't learned anything that would explain that given what was supposed to happen. I actually don't have any idea how to make sense of the whole first set of samples (#'s 2, 3 ,4) seeing as all of their bands are significantly lower than every other band on the gel.

Lanes 2, 3 and 4 look like digested plasmid, cut either twice (two fragments) or three times (three fragments). The fragments add up to about 5 kB, typical for a plasmid. Lanes 3 and 4 are identical, and lane 2 is different (with an extra restriction site). I would guess these are suspect 1, suspect 2 and crime scene sample digested with the same enzyme.

Bottom line

If this is a commercial kit, give them a call to clarify. This answer is only an explanation of the principles.

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  • $\begingroup$ That helped fill in some blanks, thanks. I think I may have got it, the sets of samples might be grouped by treatment, the treatment order is reversed, and within each treatment grouping the suspect order is reversed from what I originally assumed. where 10 is untreated crime scene, 9 is untreated suspect 1, 8 is untreated suspect 2, and then in that order for the treatments as well in the following groups of 3. But if that were the case, wouldn't lane 6 and 7 be full size linear? They are between the oc and ccc of 9 and 10 and collapse into a single band, otherwise I'm not sure what that is. $\endgroup$
    – Sidereus
    Oct 10, 2023 at 2:49
  • $\begingroup$ @Sidereus Grouping by treatment, with the crime scene between the two suspects, would make comparisons easier. Especially when the gel does not run straight. $\endgroup$
    – Karsten
    Oct 10, 2023 at 3:20
  • $\begingroup$ Sometimes you don’t see the faint second band for intact plasmid. $\endgroup$
    – Karsten
    Oct 10, 2023 at 3:21

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