Wednesday 10/16/13

Posted on October 16, 2013 by admin

10:00am – 11:00pm

Coding

  • finishing up addition of cross-correlation based drift correction for global and local region data to ChromatinCropper GUI

Mentoring

  • Bogdan finds that samples which gave extra bands on PCR failed T7 reaction. Other samples gave good (~1000ng/uL) yields.
  • A moderate fraction of the primers have quadrupole Gs (and the G7 / alpha tub that never amplifies with the T7 primer has a quintupole G).

Bogan’s first round of black regions

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

Bogdan’s current samples

  • 50 kb black domain: G08, between GRN and ANTC (try 2) – T7 failed
  • 50 kb black domain: E05, upstream of E(Pc) (try 2) – T7 failed
  • 250 kb black domain: F07, BXC_K_right, 240kb, (try 2)
  • G01, ANTC left extreme (my probe here didn’t work, not sure why)
  • F02, BXC_Y_left (15 kb) – smudgy but made T7 product.
  • F03, Ubx_through_bxd (my PCR previously worked, Bogdan’s = no PCR product)
  • F04, AbdA_through_iab4
  • F05, iab5_through_AbdB
  • F06, BXC_Y_right (50%Y 40%R 10% K) 140kb

Reorder

  • G7nT7: TAATACGACTCACTATAGGGTTGTGCGCAACGACCTAG
  • D12nT7: TAATACGACTCACTATAGGGTGTCGCGTCGGCCAGAAAC
  • E5T7: TAATACGACTCACTATAGGGCACGGCGGAGGGATAAGTTG
  • F2T7: TAATACGACTCACTATAGGGAGCGGCGTTCGACACCTTTG
  • F3nT7: TAATACGACTCACTATAGGGAGGGCGGTGCGGTACTAAG
  • F8nT7: TAATACGACTCACTATAGGGCGCGGGTCGTCCTAATAAG

PCR2 gel of Black trouble regions + new BXC regions
bogdanTroubleBlack

3 possible troubleshooting solutions

  • new primers
  • 2 rounds PCR: first without T7 to pickout library, then with T7 extended primer
  • gel extract correct size band prior to T7-IVT reaction

Loci moving forward

  • Run RNA cleanup on 50% of total T7 reaction (10 uL) –> 1,000 ng/uL yield.
  • Run RT reactions
  • Tomorrow run Oligo cleanup and diagnostic denaturing-PAGE gel.

Discussions

  • Introducing Ruobo to matlab-storm
  • fix startup_demo
  • actually merged alistair into master (checkout master then merge alistair then push master to git).

Presentation

  • Tuning up presentation for Stowers. First round through practice delivery.
This entry was posted in Summaries and tagged , , , . Bookmark the permalink.