EcoRI-HF™ shows no star activity when used in 5 minutes or overnight. FastDigest enzymes were tested to digest most of their commonly used substrates (plasmids, PCR fragments, genomic DNA) to completion in extremely short (5 minutes) time. No sequential digestions or buffer changes are needed. The 10X FastDigest Buffer is a proprietary digestion buffer which supports 100% activity of all FastDigest restriction enzymes. DNA/RNA modifying enzymes, such as ligases, phosphatases, kinases and mesophilic DNA polymerases also have 100% activity in FastDigest Buffer. Make sure to use FastDigest and HF enzymes of each, but use 2ul of HF enzyme rather then one, and increase slightly the concentration of the buffer, so like 1.1X. The FastDigest Green Buffer and Thermo Scientific FastDigest Buffer are proprietary digestion buffers which support 100% activity of all FastDigest restriction enzymes. Therefore, enzymes used in do NEB’s restriction enzyme buffer system makes your restriction digests easy and … Given that the NEBuffer 4 was the default buffer for about 2/3 of all conventional NEB restriction enzymes, it made sense to choose a universal buffer similar to NEBuffer 4 and tailor the HF enzymes to this buffer. So it would look something like this: Choose the reaction time that works for you. Locate commercially available restriction enzymes by category, name, recognition sequence, or overhang. 50 μl reactions were set up using 1 μg of Lambda DNA, 1 μl of enzyme and recommended reaction buffer. 100% activity in a single buffer. Occasionally, an enzyme has specific buffer requirements not met by one of the four standard NEBuffers, in which case the enzyme is supplied with its own unique NEBuffer. For your convenience, NEB offers over 200 Time-Saver qualified enzymes. Digests were incubated at 37°C. This system allows for double and multiple digestions with any combination of enzymes. One universal buffer enables any combination of FastDigest restriction enzymes to work simultaneously in one reaction tube and streamlines numerous downstream procedures. Over 210 restriction enzymes are 100% active in CutSmart Buffer, making it significantly easier to set up your double digest reactions.