New England Biolabs
NdeI
R0111S
Info:Supplier Page View Product Specs View Product
I routinely construct series of several plasmids for cloning and expression purposes. In such cases, there is a necessity to check constructed plasmids quickly by diagnostic restriction mapping using appropriate restriction enzymes. I have constructed a plasmid called pTS-del of size approximately 2.6 Kb, which contains NdeI sites. Restriction with NdeI will yield three DNA fragments of sizes 1246, 943, and 497 bp. Restriction of this plasmid with NdeI indeed gave correct restriction pattern as seen in 1% agarose gel analysis.
Diagnostic restriction mapping of the plasmid constructs
1 microgram of plasmid DNA
Typical restriction reactions are done at 50 microliter reaction volume with 1 microgram of total plasmid DNA, 1X final concentration of NEB's Cutsmart buffer and 1 microliter of NdeI enzyme. Reaction mix was incubated at 37 degree C for 1 hour. Then 1% agarose gel run was done to check restriction pattern.
Use correct buffer system and make sure that plasmid DNA is free of any contamination with salts.
Restriction digestion of pTS-del plasmid with NdeI gave correct diagnostic restriction pattern as seen in 1% agarose gel.
N/A
None
Easy and simple protocol
Satisfied with using NdeI restriction enzyme from NEB for diagnostic restriction mapping of the plasmids I have constructed especially pTS-del plasmid.