Established in the mid 1970's, New England Biolabs, Inc. (NEB) is the industry leader in the discovery and production of enzymes for molecular biology applications and now offers the largest selection of recombinant and native enzymes for genomic research. NEB continues to expand its product offerings into areas related to PCR, gene expression, sample preparation for next generation sequencing, synthetic biology, glycobiology, epigenetics and RNA analysis. Additionally, NEB is focused on strengthening alliances that enable new technologies to reach key market sectors, including molecular diagnostics development. New England Biolabs is a privately held company, headquartered in Ipswich, MA, and has extensive worldwide distribution through a network of exclusive distributors, agents and seven subsidiaries located in Canada, China, France, Germany, Japan, Singapore and the UK. For more information about New England Biolabs visit neb.
Restriction digests are simple. But underestimate them at your peril. Restriction digests 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. Restriction screens, where vectors are digested using restriction ezymes to generate multiple bands, are a simple and effective way to determine if your clone contains your desired insert. Vectors and inserts can contain many different restriction sites, making it confusing to select enzymes that produce interpretable results when designing a restriction screen. Add in the fact that not all restriction ezymes are equal (some can be problematic to work with) and designing a restriction screen can be a daunting task.
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 available to you (this could be your personal stocks, lab stocks, or those from another lab if you’re on friendly enough terms). Identify any potentially mischievous restriction enzymes and try to avoid them (I put an asterisk* next to them). Problematic enzymes could be those:
- that are inhibited by methylation, such as XbaI.
- that require incubation at temperatures other than 37oC. For example, BslI requires an incubation temperature of 55oC while SmaI requires an incubation temperature of 25oC. You can find details of incubation temperatures on NEB’s Restriction Enzyme Chart.
- or just enzymes that are unfriendly for whatever reason.
Step 2. Identify Sites in Your Insert
Identify restriction sites in your insert. NEBcutter V2.0 is a free and very handy tool that can be used to quickly identify restriction sites in a DNA sequence. If you can identify two sites in your insert that will produce a decent sized band following digestion 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. It is advisable to 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 Screens
Restriction digests can produce undesirable results or fail for a variety of reasons. Here is a checklist to work through if your digests are giving you surprising results or no results at all:
- the restriction enzymes have lost their activity. This could be due to age, repeated freeze thawing or inproper storage. Check each enzymes individually (if using multiple enzymes) with a positive control (such as a vector you know should give you bands).
- the restriction enzyme is blocked by methylation (some restriction enzymes are blocked when their recognition sequence is methylated).
- enzyme was incubated at an incorrect temperature. Double-check the incubation temperature for your enzyme using NEB’s Restriction Enzyme Chart.
- an incompatible buffer was used. NEB’s Restriction Enzyme Chart also provides details of compatible buffers. The activity of some ezymes can be reduced in certain buffers. When using multiple ezymes you need to ensure that all enzyme are active to an acceptable level in the buffer you are using.
- too much DNA or too much enzyme was in the reaction (this produces a ladder).
- vector or insert has a deletion.
- incomplete digestion (enzymes are not fully functional).
- a restriction site has been added or removed.
- the vector prep is dirty and may contain contaminates that inhibit the digestion reaction. Reperform the prep (e.g. miniprep) if you are concerned about this.
- too much enzyme, high ionic strength or high glycerol caused star activity (star activity is where enzymes cleave sitethat are similar, but not identical, to their standard cleavage site.
- 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. Hopefully you’ve kept your notes made when designing your restriction screen and have an idea of alternative enzyme you can use. 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 (or someone unplugged your freezer and destroyed your enzymes without you realizing).
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?
Originally published on August 11, 2008. Updated and revised on 07 January 2020.