9:45a-6:30p
Review
- first attempt at discussion written (final paragraph still needed, and abstract needs revision).
- Working on figures.
Notes for abstract / discussion
(1) There are a handful of papers that arrive at interesting inferences about stochastic gene expression using a Markov approach.
(2) There are 3 papers that actually present a general Markov framework that can be used by a non-specialist to go from a biochemical cartoon of some regulatory mechanism to an analytical expression for the moments of important gene expression properties.
(3) A substantial field exists studying just average properties through thermodynamic models. Experiments today can also the distribution of expression behaviors. The user-friendly version of the Markov approach could be applied to a host of complex metazoan regulatory architectures to understand a little more how they really differ.
In our PloS CB papers (and your PNAS paper) we layout a simple framework for going from a biochemical cartoon to a Markov model, to a calculation of moments for that model, something that is seriously lacking in previous applications of the Markov approach (e.g. Pedraza and Paulsson 2008) and has perhaps hindered its wider use by non-specialists. Your PLoS CB paper certainly does the best pedagogical job of this.
BX-C staining
- washout en probe: 2 min at 65C in 50% formamide
- set-up restain. Out of BX-C probe from Brian. Need to make more. Attempted staining with <.5 uL in 30 uL dilution.
- 15 uL not quite enough to fill chamber with 25um spacers.
Chromatin project prep
- ordered large scale Phusion PCR kit
- plate of primers moved up to rush order
- order P1 extended forward primer (for use with current secondary / tertiary scheme)
- order 405 labeled extended forward primer (for direct 405 labeling of the primary)
- order complementary labeled secondary probe (Alexa647
Primer design
% Original Design
%
% P1 seq primer probe region
% (unlabeled probe) ---------------->-------->----------->
% cy5<--------------------------------- (long secondary)
% 405-----------------> (tertiary)
%
% <--------------G-cy5 (truncated secondary)
%
designed and not used:
% tertiary + P1 + primer + probe region
% ------------------------------------>---------->------------------>
% <-----------cy5 405<-----------------
% S2a S2b
% New Design
% 2 spacer BPs
% 405 extended common / Probe
% 405 -------------------- --> ----------------->
% cy5<--------------------
% (cy5 secondary)
%
Very bad news: common primer + BsmI site has hairpin: aATTCGGcagacCCGAATgc
Which leads to dimers:
ATTCGGCAGACCCGAAT
|||||| : : ||||||
ATTCGGGTCTGCCGAAT
- this is probably death for the RT reaction at 42C. Maybe we can use shorter primers or ones that are shifted. The RNA is still going to want to self-dimerize.
- Hairpin (IDT deltaG -5 kcal/mol)
- Self-dimer -12 kcal / mol.
potential work around
- PCR amplify, (hopefully at high temperature this works)
- restriction digest to cut off bad common primer.
- Gel extract library to remove bad primer (or column purify?) Ligate on new primer