Thursday 07/18/13

9:30 A – 7:45 P, 8:30 P – 12:00 A

expanding primer sets

looking into quaternary cyclic codes to generate primers with appropriate distance
There is literature on this, but it is not obvious from the proofs and coding theory descriptions how to implement the code.

using 25mer primers as a base set of sequences from Elledge lab
converting into 20mer primers with GC clamp and Tm filter. Current parameters leaves ~80K of 240K (Tm 65-75 still a bit high for 20mers, I think the 70-80 Tm of the Jeff set also substantially limits library size).
raised Tm to the 70-80, kept 29,329 primers. This will probably still take the rest of the day and maybe night to BLAST against mouse…
Test run with first 10000 BLAST against genome: 489 have 16 or fewer hits to mouse
Fixed up a few code errors, hopefully running nicely now O/N on Mouse, Fly, Human, Monkey and E. Coli genomes.

use Zymo kit from Jeff to Gel extract
looks much better (first 2 columns P1, RC, before gel extract, second two, RC, P1 after gel extract)
still some large band — seems to be a denature/renature biproduct of the smaller bands (which is also why we get two bands again, though I just cut one).
Got T7 high RNA NEB kit from Jeff, ordered new second generation recently release kit for Jeff to replace
4.5 uL of ATP, TTP, and GTP, 3 uL of CTP plus 20 uL of CTP-cy5 = maser mix (that’s basically all my CTP-cy5).
100 nmol CTP is only like 3 reactions!
running O/N 16 hr tx reactions with 1 uL RNasin added and 10 uL new-made CTP-cy5 RNA mix.

Teaching Guisheng and Jiang about multicolor imaging with Steve and Dave software on STORM 2 and STORM 2 protocols.
set up O/N STORM on new sample round 2 (well really round 3), slide 1.
very low background, bright dots, not much switching though. probably labeled secondary no primary.
setup complete imaging session 2x
setup O/N image analysis of Gdc2 data.