Lab Aug 7 2009

1. Checked P1010-Amp and P1010-Kan transformants: Good growth on all Amp, no growth on Kan.

2. R0051(plambda cI) had no growth, and I0500(pAra) had 1 colony on each plate.

3. Both ccdB streak plates had growth.

refrigerated all plates, colonies to be picked and grown in broth culture on Monday.

All Kan plates so far have exhibited low or no growth after transformation. I'm thinking that we have a systematic problem in the way we are handling Kan transformations. The protocol states that they have to stay longer in the incubation bath before plating, but I'm not sure how consistent we've been on that. Or if there are other factors we should play with.

Given that there is only a single colony on each plate for I0050 it seems like perhaps we should grow and miniprep both of them and then do a gel to see if they are the same length. Perhaps even with a ladder so that in case they are not we can pick out the one that is what it's supposed to be.

Qiagen Miniprepped R0010 (pLac), J23032 (ribolock), J23066 (ribokey), and C0051 (λcI)

nanodropped C0051 and R0010

R0010 260/280 - 1.88, 260/230 - 2.10 = 21.2 ng/ul

C0051 260/280 - 1.65, 260/230 - 1.13 = 40.5 ng/ul

I did a second elution on both of these, one in water the other in TE.

R0010 in water - 5.9 ng/ul

C0051 in TE - 5.5 ng/ul

Unfortunately, I had assumed that since most of the other minipreps had resulted in concentrations of around 20 ng/ul that these would as well. Since the C0051 was so much more concentrated it's not really possible to do a direct comparison between water and TE in terms of elution efficiency. Next time we do minipreps I'd like to try water and TE on the same plasmid, though, because these results suggest that water might be more efficient.

plasmids stored at -20

made 20% glycerol freezer stocks for those 4 parts and placed them in the -80: Cryopreservation Protocol. (all part stocks are in a separate ziplock from our competent cells and pUC18, but on the same level of the freezer.)

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