9:30 A – 8:30P,
Probe making
- Emulsion PCR of engrailed region probe at Wu lab.
- Final library concentration 16 ng/uL, in 14 uL. Should PCR amplify once.
- Brian will run test PCR and test gel to check library quality / size distribution.
- Full process straight from original aliquot from LC not yet tested. Though directly labeled library from LC did not work.
Fly work
- Collect virgins (Espl, Sim, MTD, yw DB-hb-LacZ). Probably all a bit late, 5P. Last collected 9PM yesterday, kept at 17C O/N. Probably some non-virgins?
- Separated phenotypic virgins and potential virgins in different vials, will keep potential virgins to see if any larvae appear, then toss or cross.
- Picked up 2x copy Mat-GAL4 flies from de Pace lab.
Literature comparison
- vanO PCR based DNA FISH probes:
- ~10 Kb probe region, in 200 bp length probes
- PCR from genomic DNA with specific primers (primer amplification bias?)
- pre-screen genome for good amplicon regions / good primers
- PCR amplicons mixed, labeled with ULYSIS (sticks dyes on Gs). (http://products.invitrogen.com/ivgn/product/U21660). Might be worth trying.
- products are double stranded. (labeling reaction requires denaturation and may therefore work on single stranded probes?
- Beliveau paper
- target regions 10 Kb to multi-megabase
- single stranded probes
- Synthetic synthesis of probe sequences, emusion PCR amplified, identical matched primer removes primer bias — probably much more homogeneous set.
- fewer fluorophores per base-pair in region, but could probably do a hybrid method with ease if greater fluorophore density helps
Planning