Instructions for Restriction Analysis using the freely available Sequence Analysis tools: NEBcutter and Web Cutter

NEBcutter http://tools.neb.com/NEBcutter/index.php3

default is to cut linear DNA with all enzymes that New England Biolabs (NEB) sells

Example:
Lambda DNA can exist as a linear DNA molecule so it is OK to just select Lambda DNA ( right side of the screen) and let the program execute.  What you will get back is lambda DNA cut with all enzymes.  Most likely you are interested in what happens when you cut with one or a few enzymes.

next step:
select custom digest from lower left of the screen
you will get a new screen with a list of all the enzymes
select enzyme of choice -- for example HinDIII
scroll to find enzyme, check box then select digest from the menu along the bottom of the screen

when the next screen appears choose view gel.  This will open a new window which by default shows a computer generated image of the fragments separated on 0.7% agarose along with a size ordered list of the fragment sizes.  To view fragments separated on a different % agarose select from the pull down menu.
 

Please note that you if you are working with plasmid DNA you must be sure to select circular DNA before the initial digest in order to get more realistic results

another freely available program is Webcutter http://www.firstmarket.com/cutter/cut2.html

for this program I find it easiest to
copy and paste DNA sequence then select the proper radio buttons and look at results

open file with sequence in new browser
copy sequence only not comments on first line
paste into box, enter a title for the sequence (not required )
then select : circular sequence analysis  -- for plasmid DNA , default is linear
display : all enzymes
analysis: all enzymes or whatever subset you choose
then choose analyze this sequence

the result is a screen that shows the sequence and the site of the restriction site  followed by a table of cut sites (not sizes).
You would manually calculate the sizes by subtraction.
 
 

© 2001 Donna E. Crone, PhD, Clinical Assistant Professor, Dept of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180 croned@rpi.edu