NebCutter is a tool for analyzing DNA sequences to identify restriction enzyme sites and open reading frames (ORFs). It allows users to input sequences from various sources and customize the enzymes for digestion, displaying a schematic of the sequence with relevant information. Users can calculate the sizes of fragments produced by specific enzymes through a simple formula involving base positions.

1. NEB Cutter
(Online Restriction Digestion)
1

2. Introduction:
• NEBcutter accepts an input sequence, which can be pasted in, picked
up from a local file or retrieved from NCBI as a GenBank file via its
accession number. Various options are available to select the size of
ORFs to be displayed and the set of restriction enzymes to be used.
The program calculates the positions of all restriction enzyme sites
noting those that might potentially be blocked by overlapping
methylation and finds the ORFs in the sequence. It then displays a
schematic diagram of the sequence, the long ORFs, based on the
rules described in the Methods and all restriction enzymes that cut it
just once.
2

3. 3

4. 4

5. Custom Digest: Custom digestion involves
digesting With specific or desired enzymes.
5

6. Selection of Enzyme:
6

7. Restriction map:
7

8. Restriction sites:
8

9. Fragments Length and Position:
9

10. 10

11. On Gel:
11

12. 12

13. • How to calculate the size of fragments produced by restriction
digestion?
Subtracting the initial base number from ending base number and
then Adding one.
For example: BamHI Finds its restriction sites at 5506 bp and 22346bp
so the size of its fragment will be 16840bp as explained below:
BamHI-BamHI
5506-22346
22346-5506=16840
16840+1=16841bp
13