Timing for the big mutagenesis experiment

In the previous planning post I ended with the following breakdown:

Day -1: Streak out the cells.  (3 strains)

Day 0:  Inoculate single colonies overnight.  (3 strains)

Day 1: Dilute and grow, mutagenize, wash, dilute and grow, 

            freeze, dilute and grow, transform with NovR, wash, 

            grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)

Day 2: (Pool), dilute and grow, transform with KanR, plate.
            (6 cultures (not the controls))

Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
            plate. (6 cultures)

Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)

Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)

- See more at: http://rrresearch.fieldofscience.com/#sthash.0AuGwsEC.dpuf

Day -1: Streak out the cells.  (3 strains)
Day 0:  Inoculate single colonies overnight.  (3 strains)
Day 1: Dilute and grow, mutagenize, wash, dilute and grow,
            freeze, dilute and grow, transform with NovR, wash,
            grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)
Day 2: (Pool), dilute and grow, transform with KanR, plate.
            (6 cultures (not the controls))
Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
            plate. (6 cultures)
Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)
Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)

 I'd like to get the whole thing one as quickly as possible, because next week may be our last chance until next April to get the DNAs in for sequencing.

I have the EMS, and I've streaked out the strains, but I didn't inoculate single colonies last night.  Could I still do the big Day 1 mutagenesis, transformation and selection today, or will there not be enough time to have the cells grow up first from single colonies?

What's the actual time commitment for day 1?

1. Inoculate cells from single colonies into sBHI, grow to OD600 = 0.1.  (time = several hr)
2. Add equal volume of sBHI containing 2X the desired concentrations of EMS.  Incubate 30 min at 37°C.  Cool cells down quickly. (time = 30 min)
3. Filter-wash cultures (7 cultures) and resuspend in larger volumes of sBHI. (time = 30 min?)
4. Grow washed cells for 90 min or until OD is back to 0.1. (time = 90 min)
5. Dilute part of each culture and grow for 1 hr longer. (time = 1 hr)
6. While cells are growing, filter-concentrate and freeze the rest of the cultures.
7. Add NovR DNA fragment to cultures. Incubate 30 min, then DNase I for 5 min. (time = 40 min)
8. Filter cultures, resuspend in sBHI + novobiocin. (time = 30 min)
9. Grow (at least 6 hr or) overnight.
10. The next morning, plate some cells on Nov5 plates, freeze some, and grow some back into log phase for KanR transformation with MAP7 DNA.

OK, I think I can do steps 1-8 today.

How much EMS to use:

In #181 I calculated that adding 79.5 µl of EMS to 15 ml culture gave 0.05 mM, and 127.3 µl gave 0.08 mM.  So for my 10 ml of cells I would use 53 µl and 85 µl of EMS.

What about the volumes of culture to use?

1. Mutagenize 10 ml at OD600 = 0.1
2. Resuspend in 80 ml.
3. Freeze 70 ml after 90 min (concentrate cells first) and dilute 10 ml to 40 ml in fresh sBHI
4. Grow 1 hr, add DNA etc, filter.
5. Freeze half the cells.
6. Resuspend the rest in 100 ml + novobiocin2.5.  Grow 6 hr - overnight

Should I mutagenize a larger volume?  10 ml will be only about 3 x 10^9 cells.  In #181 (the original EMS experiment) I used 15 ml at OD600 of 0.33, which is about 5 times as many cells.

How much EMS do I have?  OK, 5 ml (after brief "Where on earth did I put it???").  It doesn't keep well once opened ("Store under inert gas") so maybe I should double the volumes for the first two steps and freeze more of the mutagenized cells for possible later analysis.  So use 106 µl and 170 µl EMS for 20ml cultures, and dilute to 160 ml.

What about the EMS-contaminated waste culture and tips?

EMS is inactivated in 1.0 M NaOH.  My filter flask will contain about 400 ml of EMS waste (including washes), so I'll add 16 g of solid NaOH to that, and let it sit for an hour before neutralization and disposal.  I'll put the contaminated tips in there too.

Day -1: Streak out the cells.  (3 strains)

Day 0:  Inoculate single colonies overnight.  (3 strains)

Day 1: Dilute and grow, mutagenize, wash, dilute and grow, 

            freeze, dilute and grow, transform with NovR, wash, 

            grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)

Day 2: (Pool), dilute and grow, transform with KanR, plate.
            (6 cultures (not the controls))

Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
            plate. (6 cultures)

Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)

Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)

- See more at: http://rrresearch.fieldofscience.com/#sthash.dxAGHC0t.dpuf

Day -1: Streak out the cells.  (3 strains)

Day 0:  Inoculate single colonies overnight.  (3 strains)

Day 1: Dilute and grow, mutagenize, wash, dilute and grow, 

            freeze, dilute and grow, transform with NovR, wash, 

            grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)

Day 2: (Pool), dilute and grow, transform with KanR, plate.
            (6 cultures (not the controls))

Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
            plate. (6 cultures)

Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)

Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)

- See more at: http://rrresearch.fieldofscience.com/#sthash.0AuGwsEC.dpuf