Lab Sep 17 2009

2.42pm

We'll be running a very large agarose gel today

1% agarose gel, 19 wells used, run at 100V for roughly an hour, visualized by developing in 0.2µg/mL ethidium bromide with shaking for ten minutes and exposing to UV light.

Contents listed from left to right, when wells at bottom of gel. (Word-wrapped for clarity)

K235009 a 	K235013 a 	K235014 a 	K235016 a 	K235017 	K115017 a 	C0012 a 	E0240 a 	R0051 a 	(Ladder) yes 	yes 	yes 	yes 	yes 	no 	no 	no 	maybe? (yes) (Ladder) 	K235009 b 	K235013 b 	K235014 b 	K235016 b 	K235017 	K115017 b 	C0012 b 	E0240 b 	R0051 b (yes) 	yes 	yes 	yes 	yes 	yes 	no 	no 	no 	no

Sept17gel.tif

7:00pm Kyliah

This looks like we haven't been miniprepping enough cells from our broth cultures - the R0051 samples are the exact same ones that failed on the September 04th gel, except this time I loaded 4µL miniprep output instead of 2µL and barely got one visible. I think we need to start double-checking the concentration of our plasmid somehow, either by checking on the absorbence reading of the overnight broth culture to ensure that it's got huuuge amounts of cells, or we need to start nanospec'ing our samples again. Maybe we can compare the "failed" samples against the obviously successful constructions in order to get a relative absorbence reading for success.

I've saved the gel for tomorrow because I don't know if I rinsed it enough or not. Maybe it should go back in the ethidium soak to get all the DNA labelled?

Mini-subcultures (1mL TE and 0.25mL broth culture) on 37C overnight:

K235009 A and B, C0012 A and B

K235013 A and B, K115017 A and B

K235014 A and B, E0240

K325016 A and B

K235017, R0051 A

Put in the waterbath by the door (no shaking alas) at 8:00pm (this volume doesn't really need too much shaking anyway, does it?)

(If this sort of culture volume is the source for today's minipreps, that may be one explanation for fails - there may not be enough cells able to grow! We really, really need to do a check of cell amount before miniprepping, or DNA concentration before running in a gel.)

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