From 2007.igem.org

Primer Selection Notes

New England Biolabs (NEB) Cutter Notes

NEB provides a NEBCutter website that allows users to freely check nucleotide sequences for restriction enzyme sites. Features include a graphical representation of sequences/plasmids with all applicable cut sites, the ability to enter in custom recognition sites for restriction enzymes, and an option to use sequences in the NCBI GenBank. Below are some brief instructions on using the NEBCutter for checking specific, user-defined restriction sites.

Procedure to use NEBCutter for checking specific restriction enzymes against a sequence submitted by Christian Ling

1. Bring up the NEBCutter page at <http://tools.neb.com/NEBcutter2/index.php>

2. Enter in your nucleotide sequence by either uploading a 'local sequence' file from your computer, inputting a
valid GenBank number, or manually entering/pasting in the nucleotide sequence into the large text box given.

3. To check the sequence against specific restriction enzymes, first click the radio button for "Only Defined
Oligonucleotide Sequences" when choosing which "Enzymes to Use".

4. Then click on the [define oligos] link. A pop-up window should appear in which multiple enzymes can be named in
the left column,and have their recognition sequence defined in the right column. Fill out all the necessary
information for the enzymes you will be using and click the 'OK' button.

5. Select whether the sequence is linear or circular and specify the minimum O.R.F length to display.

6. Name the sequence using the "Name of Sequence" text box in the middle of the page.

7. Hit "Submit" and wait for your results.