Lab Aug 28 2009

Yesterday's re-plated ligation products all grew:

k235000, K235001, K235002, K235003, K235004 and K235005

No growth in Kan broth from colony picked from the P1010 Kan plate. This is the second time. I don't believe that my colony picking technique (which has never failed up to now) could have failed twice in a row. So I'm going to have to conclude that the colonies on the P1010 plate are not viable in a Kanamycin containing broth. This would explain why we don't see a plasmid!

Lots of growth on the competent cells. Even on the 100pg plate we have lots of colonies.

Started a transformation of K235006, K235007, and K235008 from the stored ligation products. (I don't expect 5006 or 5007 to produce anything since they are on the Kan backbone that I think the broth culture test leads me to be very suspicious of. But 5008 might grow this time.) (into incubation at 12:10, out 1:30 for Kan and 2:10 for cm)

re-hydrated another P1010-Kan (plate 1, well 7K) [into incubation at 12:55, out at 2:10]

Also doing a test of competence on frozen cells from yesterday.

-Layne

I re-ran the digests from yesterday on two gels that I took a great deal of care in making up yesterday. They were both 1.5% with 50% more EtBr. Once again, we got horrible results. The bads are all messed up and the ladder is completely useless.

A couple of possibilities:

- I really suck at making gels

-We need to adjust voltage for the different concentration

-Leaving the gels in the fridge overnight is allowing water to evaporate out of them (porous matrix right? - there's definately condensation on the seran     wrap). Putting them right back into the buffer and running them right away doesn't allow for the buffer to get back into the gel and so the DNA is having to     migrate through this nonuniform mess of air and buffer.....

aug28gel1.tif

aug28gel2.tif

I stored the transformed plates from Aug 27 in the fridge as we can't do anything with them today.

Back to LAB NOTEBOOK