Chassis

Where we left off

PCR       aspC1   pTargRev   pTargetF   pcrpdt
Digest    pcrpdt  SpeI       1          spedig
Ligate    spedig                        lig
Transform lig     Mach1      Spec       pTarget-aspC1

Then, to the cells:

dsdna     donor                                    500 bp flanking aspC1, and none of it

Grow      Mach1/pCas  Kan   30           colony
Grow      colony      Kan   ara  30      pCas*     λ-Red on · Cas9 was already there
Transform pCas*  pTarget-aspC1  donor    shocked   donor is the repair template
Grow      shocked     30                 edited    no selection — the edit happens here
Grow      edited      Spec  Kan  30      picked    selects the plasmid, not the edit
Grow      picked      Kan   IPTG 30      cured     pTarget cuts itself
Grow      cured       42°C               Mach1 ΔaspC1

What happens, and in what order?