Figure 2
In vivo retrofitting of BACs a) The pRetro plasmids are cotransfected with a Cre-expression plasmid, pJM2545, into the BAC-containing E. coli. PJM2545 carries a temperature sensitive origin of replication (reptc) kanamycin resistance (KmR) and an inducible cre gene (Cre). Recombination between the loxP sites (LoxP) results in the retrofitted BACs which carry both chloramphenicol and spectinomycin resistance (CmR and SpR). b) Pulsed-field gel showing retrofitted BACs. BAC DNA was digested with NotI and separated on a pulsed-field gel. Lane 1) BACLucA, 2) BACLucARetroNeoOE, 3) BACGFPNeoOE and 4) BACGFPNeoOERetroNeoLuc. M indicates the Low Range PFG Marker from Biolabs. Sizes of the markers are indicated on the right. c) Pulsed-field gel showing BAC 4B19 retrofitted with pNELγ. DNA from BAC 4B19 (BAC) and the BAC retrofitted with pNELγ (BACNEL) was digested with NotI and separated on a pulsed-field gel. L indicates the 1 Kb DNA Ladder from GibcoBRL and M indicates the Low Range PFG Marker from Biolabs. The sizes of the markers are indicated on the right.