Thursday, August 12, 2004 , 20.40
The AvaI concatamers are subcloned into a dephosphorylated pET AvaI expression vector by bulk ligation. The ligation producrs are transformed into SURE2 (recB-A, recJ-A) supercompetent cells (Stratagene) and the colonies analyzed directly by DNA purification and restriction digestion or by PCR colony screening using Platinum Taq DNA polymerase (Gibco-BRL). This is the most critical and tedious step since bulk ligation is used. We typically screen 50-300 clones to get a concatamer of eight or more monomers.