3:00p – 7:50p
STORM
- Stop O/N STORM run of 475 kb black region F10
- spot flickering not looks dimmer
- make new Glox
- test new hybes: G3/G4 failed. F12 failed. F9/G3 failed.
- Possible reasons for failure:
- suspect extended hot wash in extra hot bath washed out probe (~60 min vs 20min).
- Probe concentrations were also a bit low (2.5 uL each).
- contamination of wash buffer to error which led to elution with the final drops of wash buffer?
Probe making
- move Bogdan’s PCRs to 4C
STORM analysis
- Running splitQVdax on F09 data
- copying F10 data to ProBox2
Data-sets needing STORM fitting
- K:\2013-10-10_F11\splitdax Now running
- H:\2013-10-07_F09\splitdax. Now running
- J:\2013-10-02_D09\splitdax (561 channel only). Now running
- J:\2013-10-03_E02\splitdax Now running
- J:\2013-10-04_G05\splitdax
- J:\2013-10-05_E06\splitdax
- H:\2013-10-09_F01V2\splitdax (not yet calibrated)
- 15 processes initialized in batch mode on Cajal. System transfer overloaded, only 5-20% processors engaged (especially while running splitdax and copying data files in the background). Hopefully things speed up once data copying and splitQVdax finish running.
Data-sets needing splitQVdax
- H:\2013-10-09_F01V2 (now running)
- K:\2013-10-11_F10
Ph project
- running simulations
- looks like reduced binding proportional to PhE/PhM and increased off-rate of PhM is too strong an effect — very little clustered observed at intermediate levels of PhM. Try affecting just cluster formation / on-rate.
- poor plotting. At low ratios of PhE to PhM most clusters contain few PhM, so PhM clustering is small. Apparently we don’t explore this regime reliably since we don’t detect this trend in the data — probably because it’s hard to tell these guys are expressing the transgene in the first place. At high ratios most clusters contain multiple PhM molecules and don’t grow easily, so clustering of PhM is again small. In between a decent cluster of PhE allows for multiple PhM molecules to co-cluster.
- changed simulation again. Now the probability of binding is given by the PhWt/totalPh ratio. If both clusters contain only Wt molecules than there are two chances. If each cluster has half Ph and half PhM then there is 1 chance. If only 1 has 50% Ph and the other 0% then there is .5 chances. The probability is determined from the number of chances as follows 1 – (1-ka)^(chances)