NUM219@NRCCAD.NRC.CA ("KEITH L. STEWARD") (03/04/90)
In a Feb 15 message posted here, John Hill <jh5f+@andrew> requested info on the efficiency of cutting multiple cloning sites with two enzymes: >One of the grad. students in our lab asked me about problems digesting >a vector with two enzymes whose recognition sites are very close >together (i.e., in a MCS = Multiple Cloning Site). I know that this >situation CAN be a problem with certain enzymes. Does anyone have a >compilation of such enzymes and their limitations? If not, does >anyone know (or is at least reasonably certain) of example(s) of this >behavior? If you want to e-mail me, I will post a compilation of the >responses. I ran into the answers the other day in an old BRL FOCUS 8:3 newsletter. An article titled 'Double Digestions of the Multiple Cloning Site' written by Joe Crouse & Doug Amorese of BRL described their analysis of the double digest efficiencies of the pUC19 MCS, and presented a table summarizing their results. I reproduce it here: First Enzyme Second Enzyme Digestion ------------------------------------------------------------- EcoR I Sst I C* Kpn I C Sma I C BamH I C Xba I C Sma I Sst I C* Kpn I N BamH I C Xba I C BamH I Kpn I C Sma I P Xba I C Sal I C* Pst I C* Pst I BamH I C* Xba I C Sal I N Sph I P Hind III P Hind III Sph I C Pst I C Sal I C Xba I C ------------------------------------------------------------ where C=complete digestion P=partial N=no digestion *=star activity evident Keith +--------------------------------------------------------------------+ | Keith L. Steward, Dept. Microbiology & Immunology, Dental Sciences | | Bldg, U. of Western Ontario, London, Ontario, Canada, N6A 5C1; | | ph. 519-679-2111 ext 6618; BITNET: NUM219@NRCCAD.NRC.CA | | +--------------------------------------------------------------------+