8:45 am – 5:50 pm, 8:45 pm – 10:50 pm
Goals
- finish letter for DF application
Coding
- small fix to accelerate FiducialDriftCorrection function
Dropbox failure
Yesterday I selectively sync’d my dropbox account on Tuck to only update the public folder and nothing else. The goal was to save space on Tuck’s harddrive. This didn’t save any space yet, it just stopped sync’ing these folders. So next I deleted the un-sync’d folders. I did this through remote-desktop connection to Tuck mediated by my primary desktop (Monet).
Today I noticed that all my files on Monet were deleted (Monet actually had dropbox uninstalled). All the files on Morgan except public were also deleted, and logging into dropbox online all the files were moved to trash despite the selective sync.
On further research, the original symptom isn’t supposed to happen: when I chose selective sync, dropbox is supposed to remove the folder from that computer immediately (not just stop synching). See this note from the dropbox help.
RNAi experiments: QPCR quantification
Well assignments Top row ‘A1-> H1’ (actually the way the label it on the plate its H1-A1, but I’ve renamed it so the numbers and letters match the spreadsheet number and letter column/row labels).
Setup
- make primer dilution and mix forward and reverse primers at a final concentration of 50 uM
- To each of 4 top left wells, add 8.4 uL corresponding cDNA and 75.6 uL of water
- move 21 uL of this mix to each of the top 4 rows.
- move 11 uL from each of the top 4 rows to the second 4 rows
- remove 9 uL from each of the second 4 rows and set aside. Add 9 uL ddH2O
- move 1 uL from each of the second 4 rows after dilution to the bottom 4 rows. Add 9 uL ddH2O
- The cDNA dilutions are now complete.
Master mix
- per well: 25 uL Phusion Master, 2.5 uL SyberGreen, 6.5 ddH2O
- 4*(12+1) wells multiplier is 52.
- 1300 uL Phusion
- 130 uL 20x SyberGreen
- 338 uL ddH2O
Final setup
- Duplicate all wells except the first row in the neighboring 4 columns.
Results:
Repeat at higher concentration
- per well:
- 12.5 uL Phusion hot start master mix
- 1.25 uL 20x EvaGreen
- 5.75 uL ddH20
- 20x master
- 250 uL Phusion hot start master mix
- 25 uL 20x EvaGreen
- 115 uL ddH20
- divide into 19.5 uL per well
- to each add well, add:
- 2.5 uL primer mix
- 3 uL cDNA
- 16 total tubes, 4 RNAi conditions and 4 gene-PCR primers
- running overnight