Lab Aug 13 2009

~9:00am Chris

Check on last night's plates:

Kan-resistant auxotroph mini-kan: lots of growth

ccdB pUC control (1hr) - colony count

ccdB pUC control (2hr) - colony count

ccdB P1010 Kan - no growth

ccdB P1010 Cm - no growth

K081005 - const. promoter +RBS (lb+amp): one double-colony...

C0083 - aspartase (lb+kan): happy colonies.

J45200-banana generator (lb+amp): 3 colonies...

J45120 - wintergreen generator (lb +amp): happy colonies

K098988 - temperature-sensitive GFP generator (lb+kan): (Kyliah asks was there no growth this morning?)

started broth cultures for J45200-banana, J45120-wintergreen, C0083-Aspartase

Put last night's broth cultures in fridge.

If growth happens on the other plates, then start subcultures.

pick up sterile epi's from central svcs

put last night's broth cultures in the fridge, cryopreserve them if you feel like it (the preserved cell lines go in the -80 on the left hand side of our shelf in the ziplock on their own - not the one with the second ziplock inside and not the one with our ccdB competent cells.)

If no growth on P1010 parts or pUC control then set the 300 ml of broth culture onto a counter at the very end of the day. (like 5pm, ideally. It needs 16 hours and I'm unlikely to be in before 9. It's in the fridge properly labelled right now.)

Could possibly run minipreps on last night's broth cultures and the couple in the fridge from yesterday. Alternatively we can run a whole bunch of minipreps tomorrow all at once.

Could also run a gel on I0500 A and B, on K145303, and R1051 (although that last one still needs to be miniprep'ed)

There is a ladder for the gel in the -20 in a white container directly under our frozen plasmids. They are the tail ends of some that Janice used for classes, but should be able to get enough for another ladder out of each (or combine leftovers from multiple tubes.)

2:00pm Kyliah

All of the no-growth plates - the DB3.1 ccdB-resistant ones and the GFP generator - seem to be showing a few small colonies now. I'm going to let them have a little more time before picking for broth culture. If no other colonies show on the K081005 plate, might either spin down remnants and re-plate, or pick and streak culture what's there on a new plate.

For the NAND gate at least, if K098988 works successfully, then we can use it as a quad-part pλcI-GFP generator, as long as we grow it between 35oC and 42oC to deactivate the on-board λcI protein.

I'll start preparing a list of what we'd like to order in stabs; the final list is going to depend on if the DB3.1 cells actually managed to get transformed on Kan and Cam plates. To order stabs, we need the part name, the plasmid it's on and its resistance, and the source plate and well.

* part BBa_I0500 - Inducible pBad/araC promoter - plasmid pSB2K3, kanamycin resistance - 2009 Kit Plate 1, well 14N or Spring 2008 Distribution, source plate 1013, well 5C * part BBa_R0051 - promoter (lambda cI regulated) - plasmid pSB1A2, ampicillin resistance - 2009 Kit Plate 1, well 6K or Spring 2009 Source Plates, source plate 2001, well 3F

5:20pm Kyliah

- Tried to pick a colony off of the P1010-Kan plate for subculturing... No idea how that's going to turn out, as we were out of wooden sticks (used the wire loop in an attempt to pick) and the colony was really, really small. Left the plate with the rest in the incubator

- Spun down and plated the remainder of K081005 in the hopes of growth without a double colony.

- Put the DB3.1 in for a 16-hour incubation with mild shaking in a water bath (set to maintain 22C minimum) at 5:25 pm - 16 hours puts that at 9:25am tomorrow. It's REALLY mild shaking - there isn't a brace taht fits this size of Erlenmayer, so I've got it kludged into place in a larger holder, and the speed really low so it doesn't pop the spring.

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