We guarantee that these products are Introduction ..........................................................................................................2 free of contaminating activities. Restriction Endonucleases Guide Our stringent quality control with the most FastDigest™ Restriction Endonucleases .........................................................2 Fermentas Restriction Endonucleases............................................................3 advanced tests guarantees you pure 1 Alphabetic List of Commercially Available Restriction Endonucleases.........6 products for your experiments. ISO9001 Recognition Specifi cities...............................................................................16 and ISO14001 is your assurance of con- Commercial Restriction Enzymes Sorted by the Type of DNA Ends Generated sistency and lot-to-lot reproducibility. Enzymes Generating 5’-protruding Ends.......................................................18 Enzymes Generating 3’-protruding Ends.......................................................18 ™ PureExtreme Quality will provide the Enzymes Generating Blunt Ends...................................................................19 performance you need for your most Enzymes Cleaving DNA on the Both Sides of Their Recognition Sequences.....19 demanding experiments. PureExtreme™ Quality PureExtreme™ Quality Guarantee..................................................................20 Activity Assay.................................................................................................20 Quality Control................................................................................................21 Storage and Shipping ....................................................................................21 Guide to Properties of Restriction Endonucleases.....................................22 Classifi cation of Restriction Endonucleases ...............................................23 Product Description Product Entry Guide.......................................................................................23 Restriction Endonucleases (descriptions of all enzymes)............................24 Nicking Enzyme............................................................................................119 Homing Enzyme ...........................................................................................120 RESTRICTION ENDONUCLEASES RESTRICTION General Properties of Restriction Endonucleases Reaction Conditions for Restriction Endonucleases General Protocol for DNA Digestion............................................................121 Five Buffer System....................................................................................121 Reaction Buffers for Restriction Endonucleases...........................................122 Dilution of Restriction Endonucleases.........................................................123 Stability During Prolonged Incubation .........................................................123 Inactivation...............................................................................................123 Considerations for Partial Digestion of DNA ................................................123 Chart: Reaction Conditions for Restriction Endonucleases...........................124 How Do I Perform a Double Digest? ............................................................127 Chart: Double Digestion using Universal Tango™ Buffer ..............................128 Activity of Mesophilic and Thermophilic Enzymes at 37°C........................130 Site Preferences by Restriction Endonucleases .........................................130 Star Activity (Relaxation of Specifi city) ......................................................131 Digestion of Methylated DNA.......................................................................132 Effect of Dam Methylation on DNA Cleavage by Restriction Enzymes............133 Effect of Dcm Methylation on DNA Cleavage by Restriction Enzymes............134 Effect of CpG Methylation on DNA Cleavage by Restriction Enzymes.............135 Effect of EcoKI and EcoBI Methylation on DNA Cleavage by Restriction Enzymes..............................................................................138 Cleavage of Restriction Targets Located in Close Vicinity within pUC19 Multiple Cloning Site....................................................................................139 Digestion of PCR Products Cleavage of PCR Products Directly After Amplifi cation .................................140 Cleavage Effi ciency Close to the Termini of PCR Products............................140 Fermentas Guide for Successful Digestions..............................................142 Information on New Cleavage Sites Newly Generated Recognition Sequences Resulting from: Removal of a 3’-overhang and Self-ligation ................................................144 Fill-in of a 5’-overhang and Self-ligation.....................................................145 Ligation of Blunt DNA Ends........................................................................148 Ligation of Protruding Compatible DNA Ends ..............................................154 1 Introduction Restriction endonucleases recognize specifi c mately 30% of all known restriction endonu- system, which combined with our extensive nucleotide sequences and cleave DNA mole- cleases. We are a leading global manufacturer quality control tests, guarantees consistent cules at a position either within or outside their of enzymes, offering 188 commercial restriction PureExtreme™ Quality – the highest quality and recognition site. These enzymes are important endonucleases. We actively screen for new performance. All Fermentas restriction endonu- tools in numerous applications, including studies restriction endonucleases and are continuously cleases are tested using the rigorous Labeled 1 of DNA primary structure and recombinant DNA discovering new restriction enzyme specifi cities. Oligonucleotide (LO) test to ensure the absence technology. More than 3600 restriction enzymes, Fermentas is the supplier of choice both for classic of even trace activities of endodeoxyribonucle- exhibiting ~260 different specifi cities, have been restriction enzymes and for new unique enzymes, ases, exodeoxyribonucleases and phosphatases. isolated. They are described in the Restriction which are not supplied by other companies. The high quality of Fermentas endonucleases Enzyme database (REBASE). Fermentas restriction endonucleases are produced makes them suitable for even the most demand- Since 1977, Fermentas has discovered approxi- under the ISO9001:2000 quality management ing applications. An Innovation from Fermentas: FastDigest™ Restriction Endonucleases Save Time – Digest DNA in Just 5min! Digestion of DNA with restriction endonucleases Features Applications can be a time consuming step in cloning and clone • Single and double digestions of plasmid DNA • Fast clone analysis. analysis. DNA digestions typically last for one hour in just 5 minutes. • Fast preparation of vectors for cloning. and often DNA is incubated with the enzymes • Enhanced performance in one hour DNA cleavage • Standard DNA cleavage reactions. overnight. The PureExtreme™ Quality of our reactions. Note enzymes has enabled us to develop a new line of • A single reaction buffer for all FastDigest™ • Low quality plasmid DNA may require longer products – the Fermentas FastDigest™ Restriction enzymes. incubation times. Endonucleases, which are specifi cally formulated • Optimal results require gel purifi cation of the to cleave DNA in just 5 minutes. 1. RESTRICTION ENDONUCLEASES RESTRICTION 1. digested DNA prior to ligation. High quality DNA is crucial for effi cient DNA diges- tion in 5 minutes. We recommend the Fermentas GeneJET™ Plasmid Miniprep Kit (#K0501) to purify DNA for FastDigest™. While FastDigest™ is ™ M1, M2 – ZipRuler Express DNA Ladder Set compatible with DNA purifi ed using kits from other (#SM1373) suppliers, Fermentas guarantees the performance C – control pUC19 DNA digested with of our products, which are tested under the most FastDigest™ EcoRI and HindIII rigorous and demanding conditions. 1-6 – miniprep DNA from recombinant clones, double digested with ™ We offer the following FastDigest Restriction M M C 1-6 – DNA cleaved with FastDigest™ REases M M FastDigest™ EcoRI and HindIII Endonucleases: 1 2 1 2 Figure 1.1. Fast clone analysis. A 2.3 kb PCR fragment was cloned into pUC19 vector. Plasmid DNA from overnight bacterial cultures of recombinant clones was purifi ed using the GeneJET™ Plasmid Miniprep Kit (#K0501) and analyzed by double digestion with FastDigest™ EcoRI and FastDigest™ HindIII (see the protocol for fast clone analysis below). Table 1.1. FastDigest™ Restriction Endonucleases. FastDigest™ Specifi city Catalog Page Analyze Your Clones 3X Faster with Fermentas Enzyme 5’ → 3’ # # ApaI GGGCC↓C ER1414 30 Protocol for Fast Clone Analysis BamHI G↓GATCC ER0054 31 ! Purify DNA from 1.5ml of overnight cultures using GeneJET™ Plasmid Miniprep Kit (#K0501). 13min BcuI (SpeI) A↓CTAGT ER1254 33 " Pipette 2µl (~0.2µg) of each miniprep DNA into thin-wall tubes. 2min ↓ BglII A GATCT ER0084 37 # Prepare the following reaction master mix: 3min BstXI CCANNNNN↓NTGG ER1024 52 Bsu15I (ClaI) AT↓CGAT ER0144 53 Water, nuclease-free (#R0581) (number of samples + 1) x 15µl Eco32I (EcoRV) GAT↓ATC ER0304 62 10X FastDigest™ Buffer (number of samples + 1) x 2µl EcoRI G↓AATTC ER0274 69 ™ HindIII A↓AGCTT ER0504 76 FastDigest