Wednesday 09/25/13

9:30a – 11:15p

STORM

• stop O/N STORM
• start copying data to Monet from O/N
• start spot-finding analysis on yesterday’s O/N splitdax F6 data.
• G2 data also not analyzed: set up DaoSTORM analysis on Cajal
• G2 bead data looks sparse — should check a few late samples and consider deleting the 561 splitdax channel to save disk space.

Probe Making

Library quality check

• Finish design and order of deep sequence adapter primers for library
• discuss design with Jeff
• recommendation: 96 well amplification PCR, take 1 uL of each and pool these together and then run adapter PCR on that.
• also take some of the independent samples directly and run separate PCRs on each.
• 

Reverse transcriptase reactions.

• take stock of RNA in 100 uL format not made into probe

Probes to try from plate (~100 kb+)

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

* all F probes have insufficient volume (e.g. BX-C genes F3 – F7).
* very low yield from 13 uL of RNA isolated on beads and resuspended in 40 uL of ddH2O
* nanodrop:

PCR

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

PCR short-regions with short primers

• E03 BLUE 13 KB (no genes, good PC/Psc)
• E04 BLUE 10 Kb (flanked by strong PolII)
• F08 RED 7 kb region next to black 1, tRNA locus
• G07 AlphaTub84 (5 kb yellow)
• G01
• G02 (positive control)
• Neg control 1
• Neg control 2

Short-regions RNA clean-up, re-numbered:

• (1) E02 13 kb green (between R/Y)
• (2) E06 Epc (12 kb YELLOW)
• (3) E07 Tou (15 kb YELLOW)
• (4) E09 RED 8 kb intronic tRNA locus, in between blue
• (5) F02 BXC_Y_left (15 kb)
• (6) F09 BLACK 18 kb region next to RED1
• (7) F12 Taf1_(17kb yellow/green)
• (8) G01 LabRegion_(Lab_Zen2) [83 kb but missing piece of ANT-C]
• All concentrations in the 1500 – 3000 (mostly ~2500) ng/uL of RNA in 40 uL.

Short-regions RT reactions: 20 uL scale

• 5uL of ~2500 ng/uL RNA. ~10 – 12 ug of RNA.
• 80*5 – 400 pmol RNA. add 300 pmol of primer.
• 5 uL RNA + 3 uL Primer + 1.5 uL dNTPs + 3.5 ddH2O
• 9x primer master = 27 Primer + 13.5 dNTPs + 18 ddH2O (8 each)
• Raw RNA (3 uL) + 3 uL primer + 2 uL dNTPs + 5 ddH2O
• 9x primer low conc master = 13.5 primer, 13.5 dNTPs, 36 uL ddH2O (7 each)
• per reaction (1 uL SSIII + 1 uL RNasin + 1 uL DTT + 4 uL FSS buffer)
• 18x master mix = 18 + 18 + 18 + 72 FSS buffer (7 each)

Prep

• Clean all RNA columns: 1x wash in .1 M Hcl (400 uL). 5x wash 1 mL H2O. 2x wash in binding buffer.

Black regions (Bogdan).

• RNA clean up. Concentration results below.
• Set up RT reactions, 40 uL scale 1.5 hours, ratio of 150 pmol primer per 5ug of RNA.

Fly work

• flipped fly stocks that didn’t get flipped on 8/26. Other stocks look pretty healthy (and current food is from 8/28). Maybe flip in a two weeks or so.