D.The Bridging PCR protocols
Bridge PCR: take normal protocol with following revision: PCR 2 bridge DNA sections with upper/midup or midlow/lower primers from model, midup and midlow primer should be designed overlap with at least 22 bp, PCR result DNA sections can be used together as models for 2nd PCR with upper/lower primers.
E.Transformation into E. coli DH5α
The ligation product was transformed into E.coli DH5α strain.
If the vector was pET28a, the strain were grown in LB plate medium containing 10μg/ml kanamycin at 37°C.
If the vector was pBAD30, the strain were grown in LB plate medium containing 100μg/ml ampicillin at 37°C.
If the vector was pSB1C3, the str ain were grown in LB plate medium containing 25μg/ml chloramphenicol at 37°C.