No matter how many times you look at it, it’s not going to change.
You are planning your next cloning experiment, but there’s a problem. The only restriction enzyme that cuts in a suitable position on your plasmid vector also, as luck would have it, cuts in another position elsewhere in the vector so you need to do a partial restriction digest to prepare your vector.
At this point I would sympathise with you. A partial restriction digest is a plasmid-cloner’s last resort. Even normal restriction digests can go wrong, but partial digests are more tricky procedures that can take a bit of time to get right.
What is a partial restriction digest?
A partial restriction digest involves performing an incomplete digestion of the plasmid DNA so that, in our example where you have two restriction sites for the enzyme in question, you will end up with three digestion products: one cut at both sites, one cut at the site you want and one cut at the site you don’t want.
Setting up the partial digest
But restriction enzymes work fast so to get a reasonable amount of partially digested plasmid, you need to play with reducing the amount of enzyme you put into the reaction, and the time of digest.
To do this you need to set up a series of digests with a fixed amount of plasmid DNA and a serial dilution of the restriction enzyme, starting with about 3-5 units of enzyme per microgram of DNA and making about 5-10 dilutions from there.
Then you heat-kill the the enzyme after exactly 10 minutes. I’s a good idea to do the reaction in the PCR machine where you can set the time and temperature accurately then go straight into the heat-kill step with no fuss.
Next you can run out the dilutions on a gel, find those that have hit the sweet spot to produce a reasonable band corresponding to the partially digested product, isolate the band and get on with the experiment.
That sounds easy, but often you have to play around with the conditions (enzyme conc/DNA conc/incubation time) to get a reasonable amount of partially digested product, using up a lot of precious time and DNA.
So I like to use a Quik alternative.
An alternative to partial digests…
I say a Quik alternative not because it takes less time, but because it involves using Stratagene’s site Quikchange site directed mutagenesis kit to destroy the unwanted restriction site with a point mutation, making a plasmid that has only the desired restriction site, which can be easily digested in the normal way.
In case you don’t know, the Quikchange procedure can be used to insert point (and longer) mutations into plasmid or linear DNA by amplifying the template DNA by PCR using primers that c0ntain the desired mutation. The procedure can be carried out quite easily on plasmids up to 10kb so is perfect for knocking out an unwanted restriction site.
The Quikchange kit contains all of the reagents you need for the procedure (apart from the primers, which can be designed using Stratagene’s primer design program), but a cheaper alternative is to do it yourself with your own reagents. This article (and video) on Jove explains how the DIY method is done and how it works.
Once you have set up the PCR reaction, the procedure is fairly hands-off. So although it will take a few more days than a partial, in my experience, the results are a lot more reliable.
Sometimes it’s better, and faster, to take the long way round.
What do you think?