I attempted to clone a small DNA insert (~100 bp after digestion) into a plasmid of unknown size, which originally carried an insert of 111 bp that I removed by double digesting the plasmid. After digesting the plasmid and the insert I wanted to incorporate into the plasmid, I ran an agarose gel to identify and purify the linearized plasmid (see picture on the left). I did not run the gel for the insert. Subsequently, I ligated the digested insert and vector using four different molar ratios of vector to insert (1:1, 1:3, 1:5, and 1:10), using 60 ng of the vector.
Analyzing the obtained results (see picture on the right), I believe that the bands indicated by the green (horizontal) arrow, present only in the lanes of the 1:1 and 1:3 ratios, likely correspond to the linearized plasmid since they exhibit slightly higher molecular weight compared to the bands in the 1:5 and 1:10 ratios. Is that right?
Questions:
1) What do the bands indicated by the orange (diagonal) arrow represent?
2) Why does the incorporation of the insert into the plasmid not increase proportionally with the quantity (in ng) of the insert? I would expect that more vector+insert complexes would form at 1:5 and 1:10 ratios.
2) What could the additional bands indicated by the question mark represent, which are quite intense in the 1:1 and 3:1 ratios but absent in the other two ratios?
You should know the size of your plasmid, you have it on an agarose gel next to a ladder.
Unless your plasmid is less that 1000 bases (not likely), you cannot visually tell whether or not it is ~100 bp larger in size on a standard agarose gel. You need a difference in product sizes of >10%.
Just proceed with transforming your cells & use sequencing to verify that your construct is correct.
Good luck!
When you say analyzing the obtained results, do you mean you just ran your ligation rxn on a gel? I agree with K.A.S.B. Transform that plasmid into cells, miniprep your plasmid, and then use purified plasmid for analysis. You can use PCR and RE digestion to help confirm before you send for sequencing. Is there a restriction site unique to one of the inserts, but not the other? Digest both plasmids with that enzyme and compare.
If you don't know the size of your plasmid (as mentioned in another comment, you have it right there next to a ladder), how did you calculate molar ratios?
More insert does not necessarily equal more ligation product. In fact, too much insert could inhibit the reaction.
Frankly, I've never ran a ligation rxn on a gel so I don't know what to expect, but if your ligation was successful, you'll have a tiny amount of circular plasmid. Idk if it would be enough to show up on a gel, that's why you need to transform it into cells, have the cells replicate the plasmid, and then isolate the plasmid (save the strain too!).
I am also assuming your gel is showing ligation products. There are going to be lots of various products from ligations, multiple inserts ligating together, multiple vectors ligating together, and all sorts of intermediate products. These tend to resolve out after transformation. So I agree with the other responders, just transform and check for correct plasmid with PCR or mini prep.
If you really wanted to understand the gel you run, you would need to have included the proper controls such as ligation with only insert and ligation with only vector.
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