Tuesday 09/17/13

Posted on September 17, 2013 by admin

Library negative controls

  • Get 10% bleach for spray bottle.
  • Bleach all pipettes. Get all new tips
  • 25 uL Phusion reactions
  • 10 uM primer dilutions
  1. Fresh library 1:100 dilution 1 uL + 1.25 uL 10uM common
  2. Fresh library 1:100 dilution 1 uL + 1.25 uL 10uM common + 1.25 uL 10 uM AbdA-T7
  3. Fresh library 1:100 dilution 1 uL + 1.25 uL 10uM common + 1.25 uL 10 uM AbdA-F
  4. Fresh library 1:100 dilution 1 uL + 1.25 uL 10uM common + 1.25 uL 10 uM E6 (non-T7)
  5. Fresh library 1:100 dilution 1 uL + 1.25 uL 10uM common + 1.25 uL 10 uM E6-T7
  6. Old library with common.
  7. Old library with common + 1.25 uL 10 uM AbdA-T7
  8. Old library with common + 1.25 uL 10 uM AbdA-F

sample 1-8 from right to left
negCntrls2

Results

  • AbdA-T7 + old library with common is contaminated.
  • AbdA-T7 with new library is clean
  • E6 +/- T7 are both clean. E6 non-T7 has no bubble band.

Setup with Bogdan

  • Overview chromatin project

PCR of black domains

  • 10 kb black domain: D09, next to Red3
  • 10 kb black domain: F09, next to RED1
  • 50 kb black domain: G08, between GRN and ANTC
  • 50 kb black domain: E05, upstream of E(Pc)
  • 100 kb black domain: F01, BXC_K_left, 125kb,
  • 250 kb black domain: F07, BXC_K_right, 240kb,
  • 250 kb black domain: F11 ANTC-L 180kb
  • 500 kb black domain: F10, 478kb region, on 3R.
  • 500 kb black domain: G10, 425 kb on X
  • AbdA-T7 negative control

mRNA project

  • designing deeper primer sets for mRNA project
  • testing length screening,21-25mer, TM 70-80: 8471, 10893, 12662, 14040, 16329
  • remove 2 out of 3 terminal GCs to 1 out of 3 terminal GCs.
  • running BLAST to genomes independently

cell culture

  • Fresh media for both vials of Kc167 cells
  • newer vial is adhering much better, should passage these soon.
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