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Restriction digests are simple. But underestimate them at your peril. Restriction digests and screens are the cornerstone of many procedures in molecular biology so getting them right, and knowing how to tell if they are going wrong, is an essential skill.
This quick guide to setting up and troubleshooting restriction screens will tell you what you need to know.
Step by step guide to designing a restriction screen
Step 1. Make a list of the available enzymes
Every lab tech and his dog has a stock of restriction enzymes available at his disposal so why not use what’s available? Write down (in alphabetical order) a list of the restriction enzymes you have. Identify any potentially mischievous restriction enzymes and try to avoid them (I put an asterisk* next to them). Problematic enzymes could be those inhibited by methylation, those that require incubation at temperatures other than 37C or just enzymes that are unfriendly for whatever reason.
Step 2. Identify sites in your insert
Identify restriction sites in your insert. If you can identify two sites in your insert that will produce a good sized band that’s great because this will increase the power of your screen by including an insert-only band.
Step 3. Identify sites in your vector
See if the restriction sites being used in the insert are also present in the vector and determine what sized bands will be produced. Then choose as many restriction sites in the vector that are required to digest the vector on the 5` and 3` sides of the MCS and around the vector. Choose restriction sites that will produce good-sized, indentifiable bands in your restriction screen.
Step 4. Perform the restriction screen
Try the screen! And hopefully it will work. If not, analyse why it might not have worked; go back to your plan and work out what sites could be present in the clone to give you the result you obtained. Perform a second screen with different enzymes to confirm the results of the first screen.
Hopefully this process will take you onto better and more informative restriction screens so that when that stumbling block does come along you can hop straight on over it and continue of your merry post-cloning way.
Troubleshooting restriction digests
Restriction digests can produce undesirable results or fail for a variety of reasons. Here is a checklist:
- the restriction enzymes have lost their activity
- the restriction enzyme is blocked by methylation (some restriction enzymes are blocked when their recognition sequence is methylated – link)
- enzyme was incubated at an incorrect temperature
- an incompatible buffer was used
- too much DNA or too much enzyme was in the reaction (this produces a ladder)
- vector or insert has a deletion
- incomplete digestion (enzymes not fully functional)
- a restriction site has been added or removed
- the vector prep is dirty
- too much enzyme, high ionic strength or high glycerol caused star activity
- the clone is actually a negative, damn
A good practice when a restriction screen fails is to setup another digest with a different set of enzymes. Much like the supervisor without morning coffee, restriction screens can be difficult to interpret at times and a second set of bands can really help work out what’s going on.
When a clone fails in a restriction screen, it’s important to question your data and be sure you know it really is a negative. Sometimes apparently negative clones are actually positive and the cloning Gods are just playing hardball.
Setting up the most informative digest possible, and going through the checklist above should help you with this and could save invaluable time and resources by avoiding either repeating the same mistakes or re-cloning the insert when you already have the clone you want!
What tips do you have for performing restriction screens?