Thursday 9/26/13

10:00a – 10:40p

Goals

• Laundry
• email Ting & Co. draft letter on progress on goals on chromatin
• Analyze existing data for new spots: density, puncta etc. scale factors? (density vs. length or something?)

Computer

• splitdax aborted in night when ProRAID ran out of disk space.
• shuffling all embryo data to Alistair10.
• Running STORM analysis on F6-405 data. Some movies launch and immediately exit and I’m not sure why…
• attempting to order new Mediasonic 8 bay harddrive enclosure with 2 4Tb Red drives. (Try the add drives as we go model rather than have one big expense. Also hard-drives seem to fluctuate in price a lot and on average decrease).

Probe making

large regions reloaded:

• Test gel for new PCRs
  1. D11 100 kb percicentric Green
  2. D12 385 kb Yellow
  3. E01 200 kb Green
  4. E8 75kb Red
  5. E10 98 kb pure yellow
  6. E12 630 kb Green
  7. F1 BXC_K_left, 125kb pure K
  8. F3 Ubx through bxd
  9. F4 AbdA through iab4
  10. F5 iab5 through AbdB
  11. F7 Bxc_K right 240 kb solid K
  12. F10 478 kb Black
  13. F11 ANTCleftBlack 180 kb
  14. G9 76 kb Blue near ANTC
  15. G10 425 kb black region
  16. Neg control 1

Gel 1 annotations:

• Loaded 1-16 top to bottom right to left, then 1-8 of 50 uL scale reactions with short primers. 2 uL PCR mix per lane.
• Very poor results. Try again with first 6, 50 uL scale.
• 50uL scale reactions with short primers worked much better.
• set up new 50 uL scale PCR with T7 extensions and .5 uL of round 1 PCR for 2-step reactions. used 2.5 uL of previous 1:20 dilutions of T7 primers for short stuff. + Neg controls 1 and 2 (abdA-NiP and abdB-NiP)
• Set up new 50 uL scale PCR reactions for large regions 1-5 + Neg control 1 (ubx-NiP)

Gel 2

• Top row: Large regions 1-5. Bottom row. small regions:
  1. E03 BLUE 13 KB (no genes, good PC/Psc)
  2. E04 BLUE 10 Kb (flanked by strong PolII)
  3. F08 RED 7 kb region next to black 1, tRNA locus
  4. G07 AlphaTub84 (5 kb yellow)
  5. G01
  6. G02 (positive control)
  7. Neg control 1
  8. Neg control 2

gel 3 annotations

• Tablet driver stopped responding again until computer reboot.
• new PCR of 6-15 (E12, F1, F3, F4, F5, F7, F10, F11, G9, G10). G10 failed. Neg controls do amplify junk again. 🙁 Fortunately this has a different size / appearance and less intensity than what’s observed in the sample channels.

RNA QC:

• Set up denaturing gel (~2 months old? qualitative inspection of gel looks good though).
• Running small regions RNA samples 1-8 + low yield RNA bead cleanup of large regions 1-5 + ladders on each end (15 lanes).
• Gel results:

  

RT QC and Purification

• small regions
• Bodgan Black regions
• Pour 4 new 15% PAGE gels.
• Run PAGE gels
• Gel1: small regions Raw RNA samples (1-8), DNA ladder, small regions Pure RNA samples (1-6),
• Gel2: small regions pure RNA samples 7-8, Black regions 1-7.
• Black black domain IDs
  • 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
    
    

Writing

• Finish first pass through trimming revision of Tx Review
• Currently 45,000 characters, down from 55,000 characters. 12.5 pages instead of 15.25 (Including figures, not including coverpage/abstract/references)