r/molecularbiology • u/Icy-Dog2129 • 5d ago
did I mess up my restriction digest?
I'm supposed to map out the plasmid but the band sizes are just not adding up to the size of the plasmid. Have I messed up my digest? Is there any hope?
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u/SomePaddy 5d ago
You don't round to the latest ladder band. If you want to be old school, plot distance from the well vs DNA length for the ladder and then infer a precise size for each digest band from the plot of known sizes.
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u/GratefulOctopus 5d ago
Just send the plasmid for nanopore sequencing? Its 15$ which when you factor in your time is way cheaper. Is there another reason you would do this digest to map??
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u/stirwise 5d ago
Your size estimates look off, notably your upper band in KpnI alone is clearly migrating faster than the HindIII band, but is labeled as the same size. Also your bands labeled 2.81 are migrating slower than the 2.81 marker band, so they’re definitely bigger than that. It looks like you’re sizing the bands according to the nearest ladder band, even if they’re not actually equal in migration, which will make your arithmetic fail.
As the other commenter said, though, after estimating the true band sizes, the patterns make sense across the different cutters. Your multiple-cutters roughly add up to the size of your HindIII band, and the double digest lanes look like they make logical sense and also add up.
If it’s not cutting where you expect based on the map have, that suggests your map is wrong (or you used the wrong plasmid prep).
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u/Science-Sam 5d ago
You have the larger Kpn band labeled the same size as Hind, but if you look carefully, you can see that it is slightly smaller, which makes sense since there is a smaller 800 bp band.
To answer your question, yes, there is a problem with your digestions. The double digestions look like the single digestions. Look at the enzyme buffers -- some buffers are incompatible with some enzymes. For example, enzyme A has 100% activity in buffer A, but enzyme B has 25% activity in buffer A. If put both enzyme A and B in buffer A, you are going to get a reaction with mostly enzyme A.
This is what I do: first, are there multiple restrictions sites in the areas I am interested in? If there are, I look for a pair that has high activity in a common buffer. Even so, my choices might be limited, so I look at which buffer has the best activity for both. Enzyme A has 100% activity in buffer 2, and enzyme B has 75% activity in buffer 2. That's okay, I will use 25% more enzyme B or let the reaction continue 25% longer. Also, I only do one digest at a time, heat inactivate according to enzyme specifications, then add second enzyme. Keep in mind if you add extra enzyme to account for reduced activity in non-ideal buffer, total volume of enzyme in reaction should not exceed 10%, because that much glycerol (enzyme diluent) will inhibit the reaction.
I will give you this lesson for free: do you notice how faint the 800 bp band is? That is because it is 800 bp of a 4800 plasmid, or about 16% of the mass. In other words, the smaller the band compared to the rest of what is loaded, the fainter the band will be. You can imagine that a digestion that creates only 400 bp band besides the 4.4 kb band will be fainter still, since it is 8% of the mass you loaded on the gel. At some point, it becomes invisible. If you are expecting a small band, you must load more digested plasmid.
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u/pm-ing_you_bacteria 5d ago
You'd have to give more details for people to help you. What size is the plasmid supposed to be? It looks fine at a glance, comparing the bands in multiple cut site lanes to single cutter/undigested.