9:15 A – 6:50P, 8:00-9:30P
- STORM analysis 750 still running, min height probably too low. Raised defaults for next time.
Prep for genotype screening
- Order primers for testing MP10 MP05 differences (seems like labeled MP10 lacks expression).
- DNA extraction using DNAzol: not able to spool DNA efficiently. PLoSONE paper Protocol recommends 2 hrs of lysis prior to ethanol addition. More ethanol slightly better yield?
- I just pelleted all the extract in ethanol. Washed with 70% ethanol, resuspend in dH2O and see if it works for sequencing.
- PCR reactions: Genomic DNA from YW, simD, Espl, each with AbdB1-F/R, simD-F/R = 6 rxns. Test on gel tomorrow
Embryo labeling
- in situs Day 2
- DFISH in situs Day 2. Fab7-488 probe from Fred works great based on first images on confocal from initial post-hybe wash.
- change labeling scheme for in situs?: Dm0-488 (conventional only, get nuclei outline for clusters-per-nucleus analysis etc.) Cy3B with low power STORM for locus #1. A647 for protein (H3K27me3 or PcGs). IR800 for locus #2? Get rid of H3-label (nice images but long time to fit and not being used very much).
Fly work
- Flies emerging from recombination crosses (hopefully we can figure out the boundaries of the Espl deletion so that the PCR screening becomes possible for recombinants).
- Ordered long taq which should help this attempt.
- Collect virgins for recombination cross
Oligiopaint probe design final step:
- cd C:\Python27
- python C:\Users\Alistair\Documents\Projects\Chromatin\Code\OligoPaints\scripts\orderFile.py -i D:\Data\Genome_TRACKS\OligoPaintProbes\BLUE_chr2R_7350000_to_7470000.bed
- F: TCCTAAATCCTAGCCCATACGGCAATG R: GGACATGGGTCAGGTAGGTTG
- python C:\Users\Alistair\Documents\Projects\Chromatin\Code\OligoPaints\scripts\orderFile.py -i D:\Data\Genome_TRACKS\OligoPaintProbes\BLACK_chr2R_15725000_to_15837000.bed
- F: GAGCAGTCACAGTCCAGAAGGGCAATG R: GTATCGTGCAAGGGTGAATGC
- python C:\Users\Alistair\Documents\Projects\Chromatin\Code\OligoPaints\scripts\orderFile.py -i D:\Data\Genome_TRACKS\OligoPaintProbes\GREEN_chr2R_400000_to_512000.bed
- F: GAGTTTGAGGCTGTGTGCTTGGCAATG R: CATAGAACGGAAGAGCGTGTG
- python C:\Users\Alistair\Documents\Projects\Chromatin\Code\OligoPaints\scripts\orderFile.py -i D:\Data\Genome_TRACKS\OligoPaintProbes\YELLOW_chr2R_3328000_to_3430000.bed
- F: GGGAGTAGGGTCCTTTGTGTGGCAATG R: TTGATCTCGCTGGATCGTTCT
- Still don’t have KaqTaq for probe synthesis.
