9:40 A — 6:20 P, 8:00 P – 11:30 P
Chromatin Paints, Library 2 finalization.
Updates to code
- fixed bug in matching region names to sub-libraries
- fixed bug in screening for nick sites.
Error checking
- spot check Drosophila sequences by using flybase BLAST of randomly selected sequences from final library to genome.
- sequence alignment should be unique
- sequences from same sub-library should map near by
- sequences should map to the same gene/chromatin-region they are labeled as
- UCSC BLAT actually does good alignment on the browser. Mouse database doesn’t do BLAST to whole genome, just genes. UCSC and Ensembl do.
Order this:
- Common-primer, RC(probe-sense-strand), RC(Nick site), RC(Index primer)
- This will give us antisense-DNA probes by either method
Probe making method 1:
- 2-5mL scale PCR with [secondary-site-extension + common-primer], [T7 + Index primer].
- (could leave off the T7. Bigger chop easier to separate on gel though).
- secondary-site-extension can be pre-labeled with 405.
- 100ug scale column clean up or EtOH precipitation.
- Nicking reaction
- EtOH precipitation
- PAGE Gel separate nicked strand
- Gel extract (butanol extraction?)
- Ethanol precipitate
Probe making method 2:
- PCR with [secondary-site-extension + common-primer], [T7 + Index primer]
- PCR column cleanup
- T7 in vitro transcribe. –> RC(secondary-site), probe-sense-strand, Nick site, Index-primer
- RNA column cleanup
- RT w/ 405-secondary site. –> 405-secondary-site,antisense-probe,Nick,Index
- stop reaction / cleanup
- ethanol precipitate and use
Library ORDERED.
STORM
- O/N PhM + Pc cy7, 647