Monday 01/07/13

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). (  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



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