Pgps3 Unique Restriction Sites _best_ «POPULAR | PLAYBOOK»

| Enzyme | Problem | |--------|---------| | | Cuts twice (once in MCS, once in the pSa origin of replication). | | EcoRV | Cuts three times (unexpectedly in the LacZ alpha fragment and the kanamycin resistance gene). | | ClaI | Cuts twice (in the MCS and the ColE1 origin). |

Just remember: no SalI, no EcoRV, and always verify your map. When in doubt, a quick virtual digest takes 30 seconds and saves three days of failed ligations. pgps3 unique restriction sites

However, successful cloning depends entirely on one thing: knowing which restriction enzymes cut once —and only once—in your plasmid. | Enzyme | Problem | |--------|---------| | |

| Restriction Enzyme | Position (approx.) | Recognition Sequence | Best Use Case | |-------------------|-------------------|----------------------|----------------| | | 1,234 | G^AATTC | Insert cloning (5' end) | | BamHI | 1,567 | G^GATCC | Insert cloning (3' end) | | HindIII | 2,345 | A^AGCTT | Backbone linearization | | XbaI | 789 | T^CTAGA | Subcloning from other vectors | | SacI | 2,890 | GAGCT^C | Terminator swaps | | PstI | 3,101 | CTGCA^G | Rare cutter for large inserts | | KpnI | 456 | GGTAC^C | Directional cloning with SacI | | NotI | 2,567 | GC^GGCCGC | For GC-rich inserts (rare cutter) | | Just remember: no SalI, no EcoRV, and