If I have lots of KanR colonies from yesterday's hypercompetence-enrichment step...

Today:

1. Pool the colonies (from each of 6 original mutagenized cultures).
2. Freeze part.
3. Make chromosomal DNA preps from the rest.  These will be used for sequencing and for the backcrosses (steps 5 & 6)
4. As controls also make DNA from the pooled NovR colonies from yesterday.
5. Use the 2 DNAs from the StrR parent and the 2 DNAs from the CmR parent to transform KW20 to StrR and to CmR.
6. Maybe use the DNAs from the wildtype parent to transform KW20 to KanR.
7. (Incidentals: make lots more BHI agar, pour plates, use frozen competent KW20, test transformation by NalR fragment, wash and autoclave flasks again.)

Tomorrow (one final round of hypercompetence selection):

1. Pool the backcross colonies derived from each of 6 original mutagenized cultures.
2. Freeze part.
3. Grow part in log phase for at least 2 hr (like yesterday)
4. Transform with a different marker (NalR fragment, or MAP7 DNA with selection for NalR or SpcR).

Monday:

1. Pool the transformant colonies (from each of 6 original mutagenized cultures).
2. Make chromosomal DNA preps from them for sequencing.