Double digests of MCS's

KEITH L. STEWARD NUM219 at NRCCAD.NRC.CA
Sat Mar 3 20:44:00 EST 1990


In a Feb 15 message posted here, John Hill <jh5f+ at 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

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 | Keith L. Steward, Dept. Microbiology & Immunology, Dental Sciences |
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