9:40a – 10:00p
Goals
- Stop O/N STORM run
- send code to Jeff
- (11a, dentist)
- Talk title for Alvaro
STORM
- Stop O/N STORM run
IVT reactions
- out of High Yield T7 buffer
- Still have 2 vials of T7 High Yield Pol Enzyme Mix though. Let’s try mixing enzyme with the Promega buffers.
Per reaction
- 8 uL 5x Promega Buffer, 4 uL DTT, 3 uL each 100mM rNTP, + 2 uL enzyme mix + 2 uL RNasin + 4 uL ddH2O + 8 template
7x Master Mix
- 54 uL 5x Promega Buffer, 27 uL DTT, 21 uL rNTP + 14 uL enzyme 14 uL RNasin + 28 uL ddH2O
- samples: D11, D12, E1, E8, E10, E12
- concentrations: 101, 105, 76, 91, 60, 30 ng/uL
pre-mixed master solution from last month
- 14 uL master + 2 uL Pol2 + 8 uL DNA template
- samples: F1, F3, F4, F5
- concentrations: 30, 10, 20, 26 ng/uL (pretty low)
Discussions
- Jeff found improved RT by Thermo: http://www.ncbi.nlm.nih.gov/pubmed/22691702.
- stats on processivity and thermal stability look better than superscript III
Ph Modeling
- current approach to handling binding is wrong. Can’t probe all clusters and just say what fraction bound, some of these didn’t just meet they were already bound and diffusing around freely.
- Really do need to keep track of two steps and to link molecules.
- should larger clusters have a higher probability of binding new molecules? (more available binding surfaces)?
- Implimented new binding algorithm. Distributions not nearly so skewed as with instantaneous binding.
- Need to do this without a loop
[v,n] = occurrences(PhP);
Allcollisions = find(n>1); % locations (x,y) from PhP which occur more than once;
Ncollide = length(Allcollisions);
PhTypes = zeros(Ncollide,2);
for nn = 1:Ncollide % loop over all new collisions
idx = ismember(PhP,v(Allcollisions(nn),:),’rows’);
PhTypes(nn,:) = sum(PhT(idx,:));
end
Advising
- Helping Hao troubleshoot bead alignment
- Fixed bead matching with proper corrmols
- still issues with data alignment in some images?