Wednesday 05/08/13

9:30 A —

Probe making

pool digests
aliquot out test DNA: 1 uL digest DNA, 3 uL TBE, 1 uL 6x loading dye + 12x gel green, 5 uL formamide with 5% 5M EDTA
Pour new 16% urea PAGE gels (2 diagonstic gels and 1 extraction gel). Will need 2 extraction gels if we are going to do all 6 samples. But maybe they won’t all have digested nicely.
Pre-run gel 1 hr.
denatured samples at 80C, 5 min. Flush wells. Load Gel. Running 1 hr in waterbath at 52 C.
column purifying and concentrating DNA samples
heating fresh elution water to 60C
CANNOT run PAGE gels with gel-green in loading buffer. DNA doesn’t move, ladder looks all wrong.
No change in number of bands before and after nicking.
If I hadn’t run the pre-digestion DNA, I would say the Ubx, AdbA and AbdB clearly all worked. Previously they gave only 1 band or two bands that were all larger than 100 bp. This is the first time I see a band under 100 bp and the first time I expect a band under 100 bp, but somehow it shows up in the un-nicked elution DNA as well!
Running another round of 1 mL PCR of Ubx, AdA and AbdB to test on denaturing gel for presence of two bands.
For Y and G the band at 100 bp in the 16% Urea-loaded gel definetely looks much stronger in the Digest than in the Elute case.
should probably gel extract these tomorrow anyway.

Brian bringing samples over ~2P.
running sample 1 O/N. Nuclei clearly visible from 647 channel. Dots also quite bright in conventional image, but not blinking much — not too much switching overall, gave up collection at 40K frames.

Looking at en data
DaoSTORM produces artifacts on dense, non-blinking data — splits into periodic structure.

* Raised threshold, test frames look like better fits.
* Re-running on Cajal O/N