10:00am – 11:00pm
- finishing up addition of cross-correlation based drift correction for global and local region data to ChromatinCropper GUI
- 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
- G7nT7: TAATACGACTCACTATAGGGTTGTGCGCAACGACCTAG
- D12nT7: TAATACGACTCACTATAGGGTGTCGCGTCGGCCAGAAAC
- E5T7: TAATACGACTCACTATAGGGCACGGCGGAGGGATAAGTTG
- F2T7: TAATACGACTCACTATAGGGAGCGGCGTTCGACACCTTTG
- F3nT7: TAATACGACTCACTATAGGGAGGGCGGTGCGGTACTAAG
- F8nT7: TAATACGACTCACTATAGGGCGCGGGTCGTCCTAATAAG
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.
- Introducing Ruobo to matlab-storm
- fix startup_demo
- actually merged alistair into master (checkout master then merge alistair then push master to git).
- Tuning up presentation for Stowers. First round through practice delivery.