Will this strategy work for verifying successful cloning of a DNA fragment into a plasmid vector?

Question

So I am in a bit of a time constraint. Essentially, I inserted a DNA fragment via molecular cloning which contains a unique RE site. I need to confirm whether my colony has or does not have the fragment. I need to do this in one day so would this work as a confirmation strategy:

1. Pick colony and perform colony PCR with primers around RE site.
2. Cleanup the PCR product and digest using the enzyme which was inserted.
3. Run on a gel. If a cut is noticed, this confirms that the colony had the inserted fragment (containing the RE site).

Would this strategy work? Are there any notable issues people can for see in this strategy?

Answer 1

Yes, your strategy would work, with the caveats already mentioned by @Cell.

Depending on which restriction enzyme and PCR buffer you use, you might even be able to skip step 2. NEB has a good list over their restriction enzymes and their activities in some common PCR buffers. With some reasoning that information is often applicable also to restriction enzymes of other brands.

Depending on your primer availability, another quick strategy is to simply use a primer pair such that one primer binds inside your fragment, and the other binds to the backbone. This should result in a PCR product only if your cloning was successful, and also verifies correct direction (in case you used blunt-end or single-site cloning). Two possible problems with this approach is:

1. If you get no bands at all you may not know if what failed was your cloning or the PCR reaction. The latter can be easily controlled for with a well-thought positive PCR control.
2. You may detect cases in which more than one copy of your fragment have been inserted. Although such cases are rare, it sometimes happens that inverted repeats of 3, 5, 7, ... etc copies get inserted in tandem.

Answer 2

One issue is if your fragment amplified is too small you may not be able to see the positive result of two bands because they run off the gel. Also the RE may not cut a small fragment efficiently.

I would recommend extracting and purifying plasmid, then do a double digest using the unique fragment cutting RE and a RE that only cuts the backbone. You should either see linearized plasmid DNA or two bands that are probably both >1 kb and easily resolvable.