Lab Aug 6 2009

9:00 am Kyliah

Prepared 2L of LB.

four plates showed growth this morning: R0010 (pLac), J23032 (ribolock), J23066 (ribokey), and C0051 (λcI) - set on to subculture

two plates did not show any growth: R0051 (pλcI, for the second time) and I0500 (pAra). Spun down the remainder from yesterday, resuspended in 100µL LB and plated again; we'll see if anything shows up.

I took a look at the ccdB entries on the distribution... A whole lot of them have inconsistent sequencing, including all of the kanamycin-resistant ones. If the two plates today don't show any growth, maybe we should order stabs of them rather than trying to retransform them; and we could order stabs of the needed ccdB lines at that time as well :S I'm not sure which would be better.

11:00 am Layne

We obtained two samples of the ccdB resistant strain from Dr. Nano's lab. The epi's only had about 60ul in each so we decided to only do two transformations with them.

We Rehydrated three backbone plasmids with death resistant genes: P1010-Amp, P1010-Kan and P1010-Chl (they all have the same part number becuse they all have the same deathn resistant gene, but the plasmids they are on have different antibiotic resistances).

We also rehydrated part J04450.

We Transformed about 60ul each of ccdB resistant cells with P1010-Amp and P1010-Kan and plated 200, 100 and 50ul of the transformants on Amp and Kan plates respectively.

We streaked the remainder of the cells from the epi's onto LB plates.

I checked the OD600 of the broth cultures started this morning to see if they were in a good state for freezing. The OD for one of the cultures was .201. The OD needs to be between .4 and about .8 to be in the optimal range for preservation, so I didn't freeze any.

For Friday:

1. Check P1010-Amp and P1010-Kan transformants

2. Check Last attempt to transform R0051(plambda cI) and I0050(pAra)

3. Check for growth on ccdB streak plates (refridgerate?)

4. Cryopreserve some of each overnight broth culture

5. Miniprep overnight broth cultures

-that's all I can think of for now..

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Stock

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21 LB + Amp plates

~30 LB + Kan plates

~30 LB plates

We may need to think about Chloramphenicol soonish.

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