5:30p – 8:30p
Bogdan / Rotation project prep:
- move black IVT reactions to top of upstairs -20C freezer
new DNA FISH in situs
- E6 (E(Pc) region, 15kb)
- F6 (Y/R 140 kb)
- Stain E6/F6 originals with 5 uL + 1 uL secondary 405-dock + 1 uL dilute 405
- Stain E6/F6 405-labeled with 5uL + 1 uL new cy5 secondary
- no primary control, cells + 1 uL new cy5 secondary
- dilute new cy5 secondary: suspend in 340 uL hybe (100 uM), dilute this 1:10 = 10 uM = 10 pmol/uL
New PCR, try 2 on all small regions with neg controls
- samples 1-8:
- E02 13 kb green (between R/Y)
- E03 BLUE 13 KB (no genes, good PC/Psc)
- E04 BLUE 10 Kb (flanked by strong PolII)
- E06 Epc (12 kb YELLOW)
- E07 Tou (15 kb YELLOW)
- E09 RED 8 kb intronic tRNA locus, in between blue
- F02 BXC_Y_left (15 kb)
- F08 RED 7 kb region next to black 1, tRNA locus
- samples 9-14:
- F09 BLACK 18 kb region next to RED1
- F12 Taf1_(17kb yellow/green)
- G01 LabRegion_(Lab_Zen2) [83 kb but missing piece of ANT-C]
- G07 AlphaTub84 (5 kb yellow)
- Neg control 1
- Neg control 2
17x master mix
- per reaction:
- 50 uL Phusion 2x master
- 1 uL of 1:20 library dilution
- 5 uL 10 uM common
- 39 uL ddH2O
- to each: add 5 uL 10 uM index primer
- Master =
- 850 uL Phusion
- 17 uL 1:20 library dilution
- 85 uL 10 uM common
- 663 ddH2O
- to each well: 95 uL